Molecular Biology Summary PDF - DNA, RNA, Genetics

Archibald Garrod (1890s) Discovery: Garrod was the first to link genetics with biochemistry. Rous Experiment: treat using RSV as a vector to deliver functional genes Miescher (nuclein-------link to hereditary), Fred Griffith Experiment: He observed that a "transforming principle" could change har...

Archibald Garrod (1890s) Discovery: Garrod was the first to link genetics with biochemistry. Rous Experiment: treat using RSV as a vector to deliver functional genes Miescher (nuclein-------link to hereditary), Fred Griffith Experiment: He observed that a "transforming principle" could change harmless rough bacteria into virulent smooth bacteria. Observation: He injected mice with different forms of the bacteria: o Virulent smooth bacteria - Mice died. o Harmless rough bacteria - Mice lived. o Heat-killed virulent smooth bacteria - Mice lived. o Heat-killed virulent smooth bacteria + live harmless rough bacteria - Mice died, and live smooth bacteria were recovered. Conclusion: This suggested that something from the dead virulent bacteria transformed the live harmless bacteria, making them virulent. It could carry inheritance (S-transforming principal) Oswald Avery This pointed towards DNA as the transforming principle. Alfred Hershey-Chase, Max Delbrück, and Salvador Luria Experiment: The "Waring Blender" experiment used bacteriophages (viruses that infect bacteria) to determine whether protein or DNA carried genetic information through a radioactive tag. Methods: o They grew E. coli in media with radioactive labels for either protein (35S) or DNA (32P). o They then infected the bacteria with phages grown in the respective radioactive media. o After infection, they used a blender to separate the phages from the bacteria. o Finally, they measured the radioactivity in the bacterial pellet and the supernatant (liquid containing the phages). Conclusion: This showed that phages inject their DNA into bacteria, proving that DNA, not protein, is the replicating genetic material. George Beadle and Edward Tatum (1958) Model organism: They used the bread mold Neurospora crassa. Findings: Most mutations affected a single metabolic pathway, suggesting that each gene is responsible for producing a single enzyme. Hypothesis: This led to the "one gene-one enzyme" hypothesis, linking genes to specific protein functions. Phoebus Levene Research: Levene investigated the chemical structure of DNA and RNA. Discoveries: He identified the components DNA and RNA are made of 4 nucleotides, mb a 2x of a structural motif (GTCA…). He also discovered the phosphodiester bond linking adjacent nucleotides between 3’C of one sugar and 5’C of another. Albrecht Kossel (1910) Contribution: Kossel investigated the chemistry of DNA. Discoveries: He identified the four nitrogenous bases in DNA: ▪ Purines: adenine, guanine – 6-membered ring focused to a 5-memebered ▪ Pyrimidines: cytosine, uracil and thymine. – single 6-membered ring ▪ Carbohydrates and phosphoric acid ▪ He also understood that complex organic molecules are built from repeating building blocks, demonstrating the concept of polymers in biological molecules. Erwin Chargaff Technique: Chargaff used chromatography to separate and quantify the different nitrogenous bases in DNA. Discovery: No. of T= A and no of C=U → "Chargaff's Rule." Rosalind Franklin Expertise: Franklin was an expert in X-ray crystallography, a technique used to determine the structure of molecules. Contribution: Her famous "Photograph 51" provided crucial evidence for the helical structure of DNA. Linus Pauling Attempt: He proposed a triple helix model of DNA with phosphate groups in the center and bases sticking out. Error: This model was flawed as it placed the negatively charged phosphate groups close together, making the structure unstable. James Watson and Francis Crick Collaboration: determine the structure of DNA. Discovery: double helix structure of DNA, with two antiparallel sugar-phosphate backbones and nitrogenous bases paired in the middle. The model explained: ○The X-ray crystallographic data. ○Chargaff's Rules (A=T and G=C). – (Cand G have triple hydrogen bond),( A and T have double hydrogen bonds). ○Heredity through the complementary base pairing allowing each strand to serve as a template for the other. Pharmacogenomics: study of how a person’s genetic makeup influences drug responses - Identify genetic variations influencing drug metabolism and drug responses - Identify genetic biomarkers Gene Therapy: to replace faulty genes to cure o Delivery system: viral vectors o Gene editing (CRISP-Cas9): precise editing of specific genes, allow for targeted corrections DNA and RNA Made of nitrogenous bases, a pentose sugar and phosphate group ▪ Lack OH at 2’ o makes DNA more stable – reduces its susceptibility to hydrolysis and enzymatic degradation ▪ OH at 2’ o Makes RNA more flexible to fold into complex structures o RNA more reactive than DNA, participate in enzymatic reactions (splicing and protein synthesis) o it has catalytic activity ▪ nucleoside: nitrogenous base and pentose sugar ▪ nucleotide: nitrogenous base and pentose sugar and phosphate group ▪ What figured out DNA structure: o Growing evidence that the base of heredity of a protein o Chargaff’s rule o X-ray crystallography o Pauling’s discovery Marshall Nirenberg / Holley / Khorana Nirenberg is best known for his work deciphering the genetic code. RNA sequence UUU coded for phenylalanine. Francis Crick determine the codon length using bacteriophages and a technique called the plaque assay. Results: o They found that only the addition or deletion of three nucleotides (or multiples of three) restored the phage's function. o This indicated that the genetic code was read in groups of three nucleotides, confirming the triplet codon hypothesis. George Gamow understanding the genetic code. Severo Ochoa the genetic code. Key Concepts and Discoveries Direction of genetic code ▪ PNPase: production of RNA oligonucleotides for genetic coding (synthesize small nucleotides) ▪ mRNA – read in the 5’ to 3’ direction ▪ start codon: AUG ▪ stop codon: UGA, UAA, UAG DNA Folding ▪ genetic functions of DNA: o informational tape to encode sequence (contain both coding info and physicochemical properties) of RNA and proteins o enable replication and thus transmission ▪ direct readout: individual bases make specific contact with a protein, between exposed chemical groups  ▪ indirect readout: binding depends on recognition of a structure, whose formation is influenced by DNA sequence ▪ DNA folding and compaction: The long DNA molecules must be condensed to fit within the nucleus while remaining accessible for cellular processes. This is achieved through a hierarchical organization involving histones, nucleosomes, and higher-order chromatin structures. DNA grooves: ▪ The major and minor grooves of the DNA double helix are crucial for DNA-protein interactions involved in replication, transcription, repair, and gene regulation. o Grooves provide accessibility for enzymes, non-catalytic proteins and other molecules to interact o Grooves contribute to DNA-protein interactions ▪ Noncovalent: HB, van der Waals and other ▪ Covalent modifications of DNA and histones ▪ Major grove is wider and carries more sequence information DNA forms: B-form A-form Z-form 10.5 bp (helical) 11 bp (helical) Right-handed Right-handed Left-handed Bp perpendicular to Bp tilted 200 with Sequences capable axis and are respect to helical of forming it are packed in the helix axis and shifted to common around center helix periphery transcription initiation sites DNA is a cylinder Grooves not as One deep, narrow deep at B-DNA groove Preferable for Stiffer than B-helix packaging ▪ Formed by stretches of alternating purines and pyrimidines ▪ B-DNA most stable ▪ At lower humidity B-DNA converts to A-form and becomes less accessible for proteins. ▪ A-DNA may form upon binding of certain proteins to DNA ▪ RNA-DNA and RNA-RNA duplexes form a thicker A-form duplex with a shorter distance btw base pairs. DNA RNA Major groove Wide and shallow Narrow and deep Type of helix A, B, and Z Only A DNA melting and renaturation: 1. DNA Replication: localized denaturation, allowing DNA pol to synthesize a new strand. 2. Transcription: DNA in the promoter →localized denaturation allows binding of txn factors, RNA pol 3. DNA Repair: the damaged DNA region is locally denatured, facilitating the recognition and repair. 4. DNA-Protein Interactions: proteins often require localized DNA melting to access specific DNA sequences or binding sites. Factors Affecting DNA Melting: G-C Content: 3 HB in G - C versus 2 HB in A -T Length of DNA Salt Concentration: High salt increase stability Temperature Denaturation is reversible. Renaturation/Annealing: A remarkable characteristic of DNA denaturation is its reversibility. When the temperature dips below the Tm (melting temp) the separated strands can spontaneously reunite, reforming the double helix. In vitro Applications: This inherent reversibility in DNA denaturation is harnessed in various laboratory techniques, including: ○ PCR (Polymerase Chain Reaction): This technique employs repeated cycles of denaturation, annealing (where primers bind to the separated strands), and extension (where DNA polymerase synthesizes new strands) to amplify specific DNA sequences. ○DNA Sequencing: Denatured DNA strands act as templates, allowing scientists to decipher the precise order of nucleotides. ○DNA Hybridization: Complementary DNA strands originating from different sources can anneal to form hybrid molecules, which find applications in various diagnostic and research endeavors. Hyperchromic Effect: dsDNA absorbs less UV light at 260 nm compared to ssDNA, because of base stacking interactions that interfere with absorbance. As DNA denatures, its absorbance increases. DNA - A Dynamic and Versatile Molecule ▪ DNA is flexible and dynamic polymer, capable of assuming various forms. This dynamism is essential for its packaging and accessibility. ▪ DNA must be compacted into small volume while maintain accessibility  Chromosome Structure: DNA molecules are very long and thin. This means that DNA must be highly compacted to fit inside the nucleus Histone proteins: These proteins assist in the compaction of DNA molecules. There are two classes of DNA-binding proteins in eukaryotic chromosomes: histones and non-histone proteins. Regular spacing of histones along the DNA. Chromatin is a complex of both classes of proteins with nuclear DNA. Nucleosomes: ▪ electron microscopy revealed that DNA is associated with DNA-binding proteins, leading to the discovery of nucleosomes, which are the basic unit of DNA packaging in eukaryotes. ▪ Nucleosomes consist of eight histone proteins (two each of H2A, H2B, H3, and H4) around which DNA is wrapped 1.7 times. ▪ H1 - helps stabilize the nucleosomes by binding btw entry and exit sites of DNA. ▪ Barrel-shaped core octamer (160bp) joined by linker DNA o DNA-histone complex is stabilized by electrostatic interactions between (+) charged a.a. of histones and (-) charged phosphate backbone of DNA. o Functions of nucleosomes: ▪ structural DNA compaction and organization, enabling DNA to fit within the nucleus. Provide structural stability and protect DNA from damage ▪ regulatory roles Critical for DNA replication, transcription, and DNA repair. Regulation of gene expression: Positioning influence the accessibility of DNA to txn factors and regulatory proteins. o Nucleosome remodeling complexes: modify position, facilitating gene activation or repression Higher-order chromatin structure: Nucleosomes and linker DNA are further compacted into higher-order structures, such as the 30nm chromatin fiber – form thru interactions btw nucleosomes and the folding of linker DNA. Further compaction: formation of higher-order structures- loops and chromosomes, enabling segregation of genetic material during cell division. Advanced microscopy techniques suggest that it may be highly irregular. DNA Supercoiling Supercoiling refers to the introduction of additional twists into the DNA double helix, deviating from its relaxed state. It can manifest as positive supercoiling (overwound/right) or negative supercoiling (underwound/left). Biological Importance: o Supercoiling enables efficient DNA compaction, allowing the extensive DNA molecule to fit within the nucleus. o negative supercoiling facilitates strand separation, easier for processes like replication and transcription. o Both make DNA more compact Under torsional stress, DNA become supercoiled with a higher intrinsic energy than relaxed DNA Both bacterial and eukaryotic nucleus, DNA usually packed as (-) supercoil. + DNA supercoil - DNA supercoil Winding direction DNA overwound in right- DNA underwound, twisting handed direction, against the natural tightening the right-handed conformation in a left- double helix handed direction Structural effects More tightly wound and Unwind facilitating compact transcription and replication Energy state Higher compared to Lower, More common relaxed RNA or DNA pol move along DNA → supercoiling Topoisomerases: - Type I Topoisomerases: o DNA relaxation o Remove supercoils during replication and transcription. o Mitosis: ▪ Resolution of DNA Knots and Tangles ▪ Separation of replicated sister chromatids o ATP-dependent enzyme ▪ 1. Bind one strand ▪ 2. Cuts it and passes an intact strand through ▪ 3. Seal the break - Type II Topoisomerases: o DNA relaxation o Mitosis: rescue entangled chromosome o DNA repair: repair of DNA ds breaks o ATP-dependent enzyme  o Cutting of one strand o Passing of a strand: the uncut DNA strand passes through the break. o Re-ligation: rejoining of the cleaved strand ▪ 1. Cut two strands ▪ 2. Passing an intact double helix through ▪ 3. Seal the break Top inhibitors as anticancer drugs o Essential role of topoisomerases in rapidly dividing cells: ▪ Crucial for DNA replication, transcription, and chromosome segregation. ▪ Cancer cells rely heavily on topoisomerase activity. o Mechanism of action: Topo inhibitors stabilize the enzyme-DNA complex, preventing the re- ligation of DNA strands leading to: ▪ Accumulation of DNA breaks ▪ Replication fork arrest o Top1 inhibitors → ss breaks o Top II inhibitors → ds breaks o Cancer cells that became resistant to Top II inhibitors become hypersensitive to Top I inhibitors Gyrases: Bacterial Topoisomerase II - Relieve the torsional stress ahead of the replication fork or during transcription - Create transient ds DNA breaks, pass one strand through the break → reseal the break. - ATP-dependent - The only known enzyme that contributes (-) supercoiling to DNA, while relaxing (+) supercoils. - Tetrameric structure composed of two A subunits and two B subunits, - DNA gyrase has a scissors’ shape: o A subunits →cutting blades (the breaking–resealing component) o B subunits → handles (the ATP-driven energy transduction component). - Because gyrase is not present in eukaryotes, potent antibiotics that block (Ciprofloxacin) gyrase activity is used to treat patients infected with a wide range of pathogenic bacteria. - Structural theme common to all topoisomerases: o Hinged clamps that open and close to bind DNA + o DNA binding cavities for storage of DNA segments. Lecture 3 Karyotype Analysis and FISH Karyotyping: Karyotype analysis involves pairing and ordering all chromosomes, providing a visual representation of an individual's chromosomes. This technique is valuable for diagnosing chromosomal disorders and studying chromosomal variations. Banding Patterns: Chromosomes exhibit characteristic banding patterns upon staining with specific dyes. These patterns reflect variations in o chromatin structure ▪ Darker bands = tightly packed chromatin. ▪ Open chromatin = lighter bands o gene expression ▪ actively transcribed genes have distinct binding patterns due to alterations in chromatin structure o base composition ▪ higher GC = darker bands ▪ higher AT = lighter bands Fluorescence In Situ Hybridization (FISH): FISH utilizes fluorescently labeled probes to identify specific DNA sequences within chromosomes. This technique allows for the detection of chromosomal translocations, rearrangements, and other abnormalities. o Chromosomal DNA is fragmented and put into bacterial cells to amplify o Probe: florescent labeled to amplify fragments o Hybridization to metaphase chromosomes Detect chromosomal translocations Centromeres: o Joins two sister chromatids o Each chromosome has a distinct centromere position, influencing the lengths of its arms and aiding in chromosome identification. o Repetitive DNA sequences that vary among diff organisms Chromosome Composition: o A chromosome is a single DNA molecule carrying an organism's genetic material and associated proteins. o A chromatid is one identical half of a duplicated chromosome. o Highly condensed: inactive heterochromatin o Less condensed: active euchromatin  o Complexity of chromosome is unrelated to chromosome number or genome size Metaphase Plate Alignment: o Metaphase: chromosomes become highly condensed and align along center of cell at the metaphase plate. This is critical for accurate segregation, ensuring each daughter cell receives a complete set of chromosomes. o Visible under light microscope. o Microtubules, attached to the centromeres of sister chromatids, generate forces that pull the chromosomes toward opposite poles for equal distribution. Centromeric DNA: o In humans, centromeres consist of repetitive alpha-satellite DNA sequences. o each chromosome has at least one higher-order repeat (HOR) arrays, with variations in monomer number and sequence conferring chromosome specificity, 97-100% identical. o Binding site for proteins involved in kinetochore assembly for chromosomal separation CENP-A: o a H3 variant, is specifically localized to centromeric regions, replacing canonical histone H3 at centromeric nucleosomes. o provides a platform for kinetochore assembly. The kinetochore: o Platform for spindle fibers attachment o is a large protein complex that mediates interactions between centromeres and spindle microtubules o involved in regulating movement of chromosomes during division genetic disorders associated with centromeres: Disruptions in centromeric DNA or CENP-A function can lead to centromere dysfunction, chromosomal instability, and genetic disorders such as infertility, Down syndrome (extra chromosome 21), and cancer (chromosome instability or tumorigenesis). Comparing Prokaryotic and Eukaryotic Genomes Feature Prokaryotes Eukaryotes Size Small (1-10 Mb) Large (3-5,000 Mb) Gene Number Small (< 10,000) Large (> 10,000) Topology Mostly circular Linear Intergenic Region Short (< 100 bp) Long (often > 100 kb) Minor component Repeat Sequence Major component (little) Pseudogenes Few Many Introns (DNA that Few more doesn’t code for proteins) Prokaryotic Genomes: Compact and Efficient Nucleoid: The bacterial chromosome is compacted into a dense irregularly structure called the nucleoid, which is not membrane-bound. Bacterial Nucleoid Nucleoid composed of a single, circular chromosome packed into a dense structure. DNA supercoiling and nucleoid-associated proteins (NAPs) play critical roles in compacting and organizing the bacterial chromosome within the nucleoid. HU Proteins: o one of the most abundant NAPs (→DNA binding proteins) in bacteria, induce bends in DNA and facilitate the formation of supercoiled loops. o Same a.a. sequence as eukaryotic H2B Plasmids: o Bacteria have plasmids, which are small, circular DNA molecules that can replicate independently of the chromosome. o Plasmids often carry genes that provide advantages to bacteria, such as antibiotic resistance or stress tolerance. o Not carry genes essential to bacterium life cycle o High copy no. = segregate randomly due to free diffusion throughout cytoplasm before cell division o Low copy no. = special plasmid partitioning system Plasmid Partitioning Systems Plasmid centromere: o specific DNA that act as binding site for partition proteins, crucial for plasmid segregation o Contain repeated sequences recognized by centromere-binding protein, ParR ParR binds to the plasmid centromere and pairs two plasmids. ParM (an ATPase or GTPase) binds ParR and forms filaments by adding ParM to the beginning of the filament that push the paired plasmids apart, ensuring their segregation into daughter cells. o ATP hydrolysis: when ParM-ATP molecules become ParM-ADP filaments depolarize Nuclear DNA Organelle DNA Organized into multiple linear Circular – no histones and chromosomes with histones and limited genes encode numerous genes Multiple non-identical High copy no. of identical chromosomes chromosomes Inherited from both parents Maternally inherited Low mutation rate High mutation rate Lynn Margulis Endosymbiont Theory: The endosymbiont theory proposes that mitochondria and chloroplasts originated from ancient bacteria that were engulfed by eukaryotic cells. Evidence for Endosymbiosis: o Genome Similarity: Organellar genomes resemble prokaryotic genomes in their size, circular structure, and gene content. o Mitochondria and chloroplasts replicate independently within the cell through - a process like binary fission, just like bacteria o Ribosome Structure: Organellar ribosomes (70S) resemble bacterial ribosomes more than eukaryotic ribosomes (80S). o Intermediate Stages Mitochondrial DNA (mtDNA): o Gene Content: Human mtDNA encodes 37 genes, primarily involved in oxidative phosphorylation and protein synthesis. o High Copy Number: Cells typically contain multiple copies of mtDNA. o Rapid Mutation Rate: mtDNA exhibits a higher mutation rate than nuclear DNA, possibly due to exposure to reactive oxygen species (ROS).  Chloroplast DNA (cpDNA): o Gene Content: cpDNA encodes about 120 genes involved in photosynthesis and protein synthesis. o Lower Mutation Rate: cpDNA has a lower mutation rate compared to mtDNA. The Central Dogma of Molecular Biology Unidirectional Flow: of genetic information from DNA to RNA to protein. DNA Replication: copying DNA to produce two identical DNA molecules, ensuring the transmission of genetic information to daughter cells. Transcription: The transfer of genetic information from DNA to intermediate molecule, mRNA Translation: The synthesis of proteins using the genetic information encoded in mRNA. DNA Replication: A Semiconservative Process Semiconservative Replication: Each newly synthesized DNA molecule consists of one original (parental) strand and one newly synthesized (daughter) strand. Meselson-Stahl Experiment: proved the semiconservative nature of DNA replication. Researchers used isotopes of nitrogen (15N and 14N) to label DNA and followed the distribution of these using density gradient centrifugation. Principle: particles when subjected to centrifugal force, will move through a density gradient until they reach a position where their density matches that of the surrounding medium. DNA Replication: Directionality and Critical Sequences Sugar-Phosphate Backbone: Nucleotides within DNA are connected via phosphodiester bonds, creating a sugar-phosphate backbone with a specific directionality. One strand runs 3' to 5', while the complementary strand runs 5' to 3'. Critical Sequences: Eukaryotic chromosomes possess three critical DNA sequences for replication and segregation: o Replication Origins: Sites where DNA replication begins - multiple. o Centromeres: Regions for spindle fiber attachment during cell division. o Telomeres: Protective structures at chromosome ends. Lecture 4 Arthur Kornberg and his son DNA Polymerase I o It polymerizes dNTPs in the 5’--->3’ direction. o DNA pol I is also involved in DNA repair, proofreading, and removal of RNA primers using 5'->3' and 3'->5' exonuclease activity + 5'->3' polymerase activity. DNA Polymerase II o can act as a backup for DNA Polymerase III during replication. o Both: DNA replication (5′ -> 3′ DNA synthesis; high fidelity) + proof- reading: 3’-> 5’ exonuclease activity DNA Polymerase III o DNA pol III is the main DNA builder. o It has high speed and processivity and is involved in DNA replication (5′ -> 3′ DNA synthesis) and proof-reading (3'-> 5' exonuclease activity). Polymerase Chain Reaction (PCR) o This technique utilizes DNA polymerase from Thermus aquaticus o T. aquaticus grows best at approximately 70 °C, making its DNA polymerase thermostable, with an optimum temperature of 72°C, and resistant to denaturation at 95 °C. o The T. aquaticus DNA polymerase is also highly processive. o Lacks 3'-> 5' exonuclease proofreading activity Bi-directional replication fork o The replication fork moves in two directions starting from the ori. o A multienzyme complex is involved in the DNA synthesis of two new daughter strands, the leading strand and lagging strand. RNA primers:  DNA polymerase cannot initiate synthesis independently; it requires a pre- existing primer with a free OH group to extend. RNA primers, synthesized by the enzyme primase (can initiate RNA synthesis de novo), are utilized on both the leading and lagging strands of DNA. o These primers are relatively short and are complementary to the DNA template strand. o Leading strand = one primer o Lagging strand = many primers → Okazaki Fragments DNA polymerase α o Pri 1 is the catalytic subunit of primase. o Pri 2 is the regulatory subunit of primase. o DNA polymerase I possesses limited processivity and extends the RNA primer. Notably, it lacks 3′ exo proofreading activity. o Initiating DNA synthesis on the leading and discontinuous Okazaki fragments on the lagging strand DNA polymerase δ comprises 4 subunits and is involved in: o Synthesis of the leading strand during DNA replication by elongating RNA- primed Okazaki fragments o DNA synthesis 5’ →3’ o DNA repair 3’ →5’ exp o moderate processivity DNA polymerase ε o Synthesis of leading strand during DNA replication o DNA synthesis o DNA repair o More processive and accurate Leading strand synthesis o Prokaryotes = DNA pol III o Eukaryotes = DNA pol ε Lagging strand synthesis o Prokaryotes = DNA polymerases I and III, with DNA polymerase I also removing and replacing RNA primers with DNA. o eukaryotes = DNA polymerase α initiating replication by extending RNA primers, while DNA polymerase δ extends Okazaki fragments. DNA polymerase γ o In mitochondria o Replication and repair of mitochondrial DNA o DNA and exhibits 3′→5′ exonuclease activity for proofreading.  DNA polymerase IA and DNA polymerase IB o In chloroplasts o Similar to bacterial DNA polymerase I. o DNA polymerase IB contributes to DNA repair. DNA polymerase processivity: Most DNA polymerases tend to synthesize only short DNA sequences before detaching from the DNA template. But need to stay tight on leading strand. Processivity refers to the ability of a DNA polymerase to remain attached to the DNA template and synthesize a long continuous strand of DNA. It allows a DNA pol that has just finished one Okazaki fragment to be recycled quickly and begin working on the next Okazaki fragment Sliding clamp: known as Proliferating Cell Nuclear Antigen (PCNA), serves as S phase marker homotrimer encircles DNA and slides along duplex keeps DNA polymerase securely bound to the DNA template. It releases DNA pol when it runs into ds region of DNA Clamp loader: Replication Factor C (RFC) is an ATP-driven multiprotein machine that couples ATP hydrolysis to the opening and closing of PCNA. RFC binds ATP, followed by binding to PCNA and DNA, leading to the opening of the clamp. Inserts primed DNA through PCNA o DNA binding triggers ATP hydrolysis, causing the closure of the PCNA clamp. Helicase - ATP-dependent molecular motors: Binding: Helicases exhibit sequence and structure specificity in binding. o Replication helicases frequently bind to replication origins o DNA repair helicases target damaged DNA. ATP Hydrolysis: energy required to separate the two DNA strands. Unwinding: move along the DNA strand, disrupting hydrogen bonds and separating the strands. Product Release: release the single-stranded DNA or RNA, preparing for another cycle. two types: ▪ DNA helicases = DNA replication, repair, and recombination. ▪ RNA helicases = RNA transcription, splicing, and translation.  In eukaryotes → slower than prokaryotes and they move along the leading strand of DNA in the 3’ → 5' direction. Eukaryotic replicative helicase CMG (Cdc45, Mcm2–7, GINS). Ring-shaped hexameric helicases in replication Mcm2–7, look like two stacked rings, has dumbbell-shaped subunits with N- and C-terminal domains (NTD and CTD). o The CTD has ATP sites and acts as a motor, driving translocation and DNA unwinding. During the G1 phase, the inactive Mcm2–7 hetero-hexamer is loaded onto DNA S phase: Mcm2–7 is activated by cell-cycle kinases to form the active CMG helicases CMG unwinds DNA through strand-exclusion, encircling and translocating along one strand while excluding the other. Single-strand DNA-binding (SSB) proteins: The major SSB Replication Protein A (RPA), bind tightly and cooperatively to exposed single-stranded DNA. Aids helicase in stabilizing the unwound, single-stranded conformation, preventing the formation of short hairpin helices. They also protect DNA from degradation and prevent secondary structure formation. Bind tightly and cooperatively to exposed DNA Don’t cover bases, which remain available as templates polymerase α-primase: It synthesizes an RNA primer followed by a DNA extension The newly synthesized RNA/DNA primer provides a free 3'-OH group as the starting point for DNA polymerase δ or ε to extend the primer. RNA primer removal: In prokaryotes, DNA polymerase I removes RNA primers using its 5′ → 3′ exonuclease activity. It fills the resulting gaps with DNA nucleotides, a process called "nick translation." Once RNA primer is replaced with DNA, DNA ligase seals the nicks btw the Okazaki fragments to create continuous DNA. In eukaryotes, the short flap pathway is employed. o Flap endonuclease 1 (FEN1) directly cleaves short RNA flaps, removing the RNA primer. Replisome, a complex responsible for DNA replication: components of the replisome: o Helicase: Unwinds the DNA double helix o Primase: Synthesizes RNA primers o DNA polymerases (in eukaryotes): DNA polymerase ε for the leading strand and DNA polymerase δ for the lagging strand o Sliding clamps and clamp loaders: Ensure the processivity of DNA polymerase o Single-strand binding proteins (RPA): Stabilize the unwound DNA o Topoisomerase: Relieves torsional strain ahead of the replication fork o Nuclease: Removes RNA primers o DNA ligase: Seals nicks between Okazaki fragments on the lagging strand o CMG helicase - central to the assembly of the replisome and its association with replication forks through entire elongation phase. DNA polymerase requires a primer, while RNA polymerase does not: RNA polymerase does not need efficient proofreading because errors in RNA synthesis are not inherited and have no long-term consequences. RNA polymerases can initiate new polynucleotide chains without a primer. Replication fork process: Step 1: Replication Fork Formation: o Helicase unwinds the DNA double helix into two single strands: the leading strand (3’ -> 5’) and the lagging strand (5’ ->3’). o Single-strand DNA-binding (SSB) proteins coat the DNA strands to prevent degradation and secondary structure formation. o DNA topoisomerases prevent DNA tangling during replication. Step 2: Priming: o DNA polymerase α synthesizes an RNA primer, followed by a DNA extension. Step 3: Elongation: Clamp loader (RFC) help PCNA to load pol δ/ε. Step 4: Primer Removal and DNA Ligation: o Nucleases, specifically DNA polymerase I in prokaryotes and FEN1 (Flap endonuclease 1) in eukaryotes, remove primers. o Ligase – ligates nicks Chromosomes: the centromere, two telomeres, and replication origins: Carrying genes, acting as the "information tape" Faithful replication Separation and partitioning into daughter cells Bacteria, which have circular DNA molecules. However, some microorganisms, possess linear genomes. Linear DNA molecules have free ends, which must be distinguished from DNA breaks. Therefore, they require terminal structures similar to eukaryotic telomeres. In Borrelia and Agrobacterium, the chromosome ends are covalently linked between the 5′- and 3′-ends of the DNA double helix. replication process in Borrelia: Replication begins internally and proceeds bi-directionally. As replication reaches the ends, replicated telomere junctions are formed. Telomere resolvase cleaves and rejoins DNA ends, resulting in the separation of replicated DNA into two linear daughter molecules with covalently closed hairpin ends. problem of replicating the 3’ ends of the lagging and leading strands: When the replication fork reaches the end of the chromosome, the final RNA primer on the lagging strand and the leading strand cannot be replaced by DNA because there is no free 3ʹ-OH end available. This inability to fill the gap leads to 3’ end shortening with each round of replication. Telomeres: specialized repeated nucleotide sequences at the ends of chromosomes. In humans, the GGGTTA unit is repeated approximately 1,000 times at each telomere. Telomeres interact with the enzyme telomerase, which replenishes the 3’- ends, and the protein complex shelterin, which protects the ends from being "repaired." Nucleases "chew on" the 5' end of telomeres, creating a 3’ single-stranded overhang. This overhang, along with the double-stranded GGGTTA repeats, attracts a six- subunit protein complex called shelterin, forming a protective cap. Shelterin: Telomeric Repeat-binding Factors 1 and 2 (TRF1, 2) are the major telomere- binding proteins that bind to the double-stranded DNA. Knocking down TRF1 or 2 leads to genomic instability and chromosome end fusion. Protection Of Telomeres 1 (POT1) binds to the single-stranded DNA.  It "hides" telomeres from the DNA damage detectors This prevents inappropriate repair attempts that could be detrimental to the cell. Brings in telomerase Telomerase, which consists of two main components: TERC: A long non-coding RNA that serves as a template for synthesizing telomeric DNA repeats. In humans, telomere DNA is composed of TTAGGG repeats, which are complementary to the AAUCCC sequence in TERC RNA. TERT (Telomerase Reverse Transcriptase): The catalytic subunit with RNA-dependent DNA polymerase (reverse transcriptase) activity. It synthesizes DNA using RNA as a template. telomerase binding and extension: Telomerase binds to the 3’ single-stranded overhang at the 3’ end of the chromosome. Using its RNA template (TERC), telomerase adds DNA nucleotides to the 3' end of the single-stranded overhang, extending the telomere. DNA polymerase α, which includes a DNA primase subunit, completes DNA strand synthesis at telomeres by extending the RNA primer laid down by primase. Telomere length shortening: In most dividing cells, telomerase activity ceases, leading to the gradual shortening of telomeres. Cells lose 100–200 nucleotides from their telomeres every time they divide. After numerous cell generations, critically shortened telomeres trigger a DNA damage response, leading to permanent withdrawal from the cell cycle, a state known as replicative cell senescence. relationship between telomeres and aging: Reactivating telomerase can lead to cell immortalization. Genomic instability: Critically short telomeres are recognized as DNA damage, which can result in inappropriate repair and chromosomal rearrangements. Shortened or dysfunctional telomeres can lead to: o Chromosome end-to-end fusions o Genomic instability o Increased risk of tumorigenesis Telomerase is crucial for stem cells and other highly proliferative cells.  Cancer: Reactivating telomerase is a common characteristic of many cancers, allowing cancer cells to divide uncontrollably and avoid cell death. This contributes to tumor growth and progression. Lecture 5 Prokaryotic DNA Replication Origin of Replication: Prokaryotic DNA replication starts at a single origin of replication (ori), which is typically a short A-T rich sequence. - 2 replication forks move in opposite directions until all the DNA is replicated. New round starts before previous has finished. - 2 newly synthesized circular DNA molecules are catenated forming interlinked rings which then need to be untangles - Untangled by DNA Gyrase (Top II), which binds to catenated DNA and introduces ds breaks to separate the rings and then it is sealed. Initiation: The initiator protein DnaA (highly conserved in all bacteria), in its ATP- bound active form, binds to the ori. DnaA-ATP destabilizes the DNA adjacent double helix, allowing for the recruitment of DnaB helicase. DnaB: the major replicative helicase in E. coli o Belongs to hexameric DNA helicase family. o Placed around ssDNA o Functional enzyme that: ▪ DNA unwinding – moves in 5’ →3’ direction – central channel (+) ▪ ATP hydrolysis ▪ Loads DnaG primase for primer synthesis to allow for replication Helicase Loading: DnaB helicase is loaded onto the ssDNA with the help of DnaA. Primase Activity: DnaG primase synthesizes RNA primers on the ssDNA. It is recruited by DnaB helicase. DnaG loads at the N-terminal domain (NTD) which causes a conformational change → get activation of ATPase in CTD Elongation: DNA polymerase III is the primary polymerase involved in DNA synthesis. Tag of War: DnaB helicase moves in the 5'→3' direction, while DnaG primase moves in the 3'→5' direction but synthesizes in the 5’ -> 3’ direction. This opposing movement creates a "tag of war," which is resolved by DnaB pulling an ssDNA loop, allowing DnaG to initiate primer synthesis. Termination: The two replication forks meet, and DNA replication ends. The two resulting circular DNA molecules are initially catenated, forming interlinked rings.  Decatenation: DNA gyrase (a type II topoisomerase) separates the interlinked DNA molecules by introducing double-strand breaks, passing one DNA duplex through the other, and resealing the breaks. Regulation of Prokaryotic DNA Replication are sufficient nutrients for a complete round of replication. DnaA Regulation: DnaA activity is regulated by ATP hydrolysis. DnaA-ATP is active, while DnaA-ADP is inactive. The duplication of DnaA recognition sites in the genome titrates free DnaA, reducing its availability. Duplication of specialized chromosomal regions (DARS) stimulates re-charging – ADP → ATP. Ori Regulation: The ori is also regulated to prevent immediate re-initiation. After replication starts, the ori enters a refractory period during which it is inactive. This period allows time for mismatch repair. The ori becomes active again when all adenine bases within it are methylated, and the initiator proteins are restored to their ATP-bound states. Inactive = hemi-methylated. Active = methylated Eukaryotic DNA Replication Multiple Ori: Eukaryotic chromosomes have multiple origins of replication to ensure that the large genome can be replicated rapidly. Slower Replication Rate: Eukaryotic replication forks move much slower than prokaryotic forks. This difference in speed is partly due to the challenges of replicating DNA packaged in chromatin. Replication forks operate independently on each chromosome Pre-Replication Complex (pre-RC) The pre-RC assembles at the ori during the G1 phase of the cell cycle to prepare for DNA replication. Assembly: The origin recognition complex (ORC), a six- subunit complex, binds to the ori and acts as a platform for recruiting other factors. Pre-RC: Cdc6 and Cdt1, the "helicase loaders," are recruited by the ORC. Mature–RC → ORC+Cdc6/Cdt1 load DNA Helicase: MCM Phosphorylated ORC can’t accept helicases (inactive) In G1, CDK2 kinase activity is low → ORC is active S phase: helicases open double helix to load remaining proteins Regulation: The formation of the pre-RC is regulated by cyclin-dependent kinases (CDKs). CDK activity is low in G1, allowing the ORC to be active and the pre-RC to assemble. In S phase, CDK activity increases. CDK2 phosphorylates Cdc6 and Cdt1, inactivating them and preventing further helicase  loading. This mechanism ensures that each ori is activated only once per cell cycle. Phosphorylated Cdc6 → degraded Phosphorylated Cdt1 → inactive Chromatin Remodeling Eukaryotic DNA is packaged into chromatin, which must be remodeled to allow the replication machinery access to the DNA. Nucleosome Eviction: Nucleosomes, the basic unit of chromatin, must be removed or displaced ahead of the replication fork, aided by chromatin remodeling complexes. Chromatin Reassembly: Following replication, chromatin must be reassembled behind the replication fork. Parental histones are distributed to the daughter DNA molecules, along with newly synthesized histones. Histone chaperones help. As a replication fork moves forward, histones are transiently displaced. Efficient replication required to destabilize the DNA–histone interfaces: o Chromatin structure is relaxed by the removal of the linker histone H1 (stabilizes nucleosomes by binding between the entry and exit sites) Chromatin remodelers: large, multiprotein, ATP-dependent complexes that restructure nucleosomes Nucelosome removal: - Sequential removal of H2A/H2B dimers and of H3/H4 tetramer OR entire octamer is replaced. - Dimer = H2A-H2B (half old; half new) Preferred octamer = - Dimer = H3-H4 (H3/H4)2 and H2B-H2A - Tetramer (H3/H4)2 – loaded first Reformation of Chromatin Behind the Replication Fork Following replication, chromatin consists of 50% parental histones and 50% newly synthesized histones. Here’s how the process occurs: The old histone octamer breaks down into an H3/H4 tetramer and two H2A-H2B dimers. H3/H4 tetramers (both old and new) are randomly distributed to the daughter DNA strands. H2A-H2B dimers (a mix of old and new) are randomly added to complete the nucleosomes. The stepwise process starts with the H3/H4 tetramer loading, followed by the deposition of two H2A-H2B heterodimers. Histone Chaperones: - Tight interaction: nucleosome with DNA - Histones are highly basic proteins - Nucleosome (+) charges of histones is neutralized by (-) of P in DNA phosphodiester backbone = tight DNA/nucleosome interaction - Histones free from chromatin need help of chaperone acidic proteins (-) to prevent aberrant aggregation. New Histone Protein Synthesis The length of Okazaki fragments in eukaryotes is determined by the point where DNA polymerase δ on the lagging strand encounters a nucleosome. During the S phase, the demand for histones increases dramatically to accommodate the newly replicated DNA. Cells synthesize a large number of new histone proteins. Most eukaryotes have multiple copies of histone genes to ensure adequate production. Histone synthesis is coupled with DNA replication Histone mRNA levels rise due to increased transcription and decreased mRNA degradation. Interestingly, histone proteins are very stable compared to their mRNA counterparts. Time Matters The cell cycle can be divided into two main phases: Interphase: This phase, encompassing G1, S, and G2, occupies around 90% of the cell cycle. o The majority of cells (80-90%) are in the G0 phase, a quiescent state within G1. o G1 typically lasts for 6-12 hours. o S phase, where DNA replication occurs, takes about 6-8 hours. o G2 phase lasts for 3-6 hours. M phase: This phase, involving mitosis (division of the nucleus) and cytokinesis, accounts for the remaining 10% of the cell cycle. It usually takes between 30 minutes and an hour. Centrosome Interphase: responsible for creating a microtubule array Mitosis: assist in bipolar spindle assembly Before cell division: the centrosome duplicates. As division begins, the two centrosomes migrate to opposite poles of the cell acts as the major microtubule-organizing center in the cell. Centrosome Structure and Composition The centrosome, a non-membrane bound organelle is the primary microtubule- organizing center (MTOC) in cells. It consists of two centrioles, arranged perpendicularly to each other, surrounded by a protein-rich pericentriolar material (PCM). Centrioles are barrel-shaped structures made of nine microtubule triplets. The PCM is responsible for concentrating tubulin and nucleating microtubules, acting as the MTOC. It also serves as a platform for protein complexes that regulate organelle trafficking and spindle assembly. Centrosome Functions The centrosome plays a crucial role in various cellular functions, including: Cell Motility Cell Polarity Maintenance of Cell Shape Cell Division Distribution of Cell Organelles such as mitochondria, Golgi, and other vesicles through its microtubule-organizing capabilities. 2 centrosomal complexes o Microtubule nucleating complex o Microtubule anchoring complex Centrosome Cycle The centrosome duplicates once per cell cycle in a highly regulated process. G1 Phase: A single centrosome with a pair of centrioles sits near the nucleus and nucleates an array of interphase microtubules. S Phase: The centrosome duplicates synchronously with DNA replication. G2 Phase: The duplicated centrosomes separate and migrate towards opposite poles of the cell. Prophase: The centrosomes reach the opposite poles, establishing the bipolar spindle. Global Reorganization During G2/M Transition The transition from the G2 phase to the M phase (mitosis) involves a significant reorganization of cellular architecture: Cytoplasmic Changes: o Mitotic Cell Rounding occurs due to the temporary loss of cellular adhesion. o Cytoskeletal Reshaping involves the assembly of the actomyosin cortex. Nuclear Modifications: o Nuclear Pore Complex (NPC) Disassembly o Nuclear Lamina Depolymerization  o Nuclear Envelope Permeabilization allows microtubules to access chromosomes and assemble the mitotic spindle. These changes are driven by increased cyclin-dependent kinase activity, ensuring coordination between cytoplasmic and nuclear events. Mitotic Cell Rounding Happens within a few minutes at the onset of the M phase. Provides symmetry that facilitates the partitioning of organelles. In interphase, cells attach to the extracellular matrix through integrin-containing focal adhesion (FA) complexes. At the beginning of mitosis, Cyclin B1-CDK1 activity causes FA disassembly. Stages of Mitosis Mitosis involves several distinct stages: 1. Prophase: o Chromatin condenses into discrete chromosomes, each consisting of two identical sister chromatids joined at the centromere. o The mitotic spindle forms between the two centrosomes. o The nuclear envelope breaks down. 2. Metaphase: o Centrosomes are positioned at opposite poles of the cell. o Chromosomes align along the metaphase plate at the cell's equator. o Spindle fibers from each pole extend towards the metaphase plate. o Each chromatid assembles a kinetochore at its centromere. Kinetochores of sister chromatids face opposite poles. o Chromatids attach to kinetochore fibers emanating from opposite poles. 3. Anaphase: o Sister chromatids separate into individual chromosomes and move towards opposite poles due to microtubule depolymerization at the kinetochore end. o Chromosomes move centromere-first, forming a V-shape. o Cell poles move further apart, elongating the cell. 4. Telophase: o Non-kinetochore microtubules further elongate the cell. o Daughter nuclei start forming around the chromosomes from fragments of the parent cell's nuclear envelope. o Chromatin uncoils, and chromosomes become less distinct. o The cell membrane starts to cleave in preparation for cytokinesis. Nuclear Envelope Remodeling During Mitosis During mitosis, spindle microtubules need to interact with kinetochores. - facilitated by nuclear envelope remodeling, which can occur in two main ways: Open Mitosis: The nuclear envelope completely disassembles, allowing spindle microtubules direct access to chromosomes. Closed Mitosis (Yeast): The nuclear envelope remains largely intact, with spindle microtubules extending through nuclear pores to reach the chromosomes. Most eukaryotes employ a process that falls somewhere between these two extremes. Spindle Shortening During Metaphase Microtubules are hollow tubes composed of α and β tubulin dimers. They have a plus (+) end and a minus (-) end. The minus end is more stable and depolymerizes slower than the plus end. During metaphase, microtubules primarily depolymerize at their plus ends while staying attached to kinetochores. This depolymerization generates the force that pulls chromosomes towards the spindle poles. Spindle Orientation in Epithelia Epithelial cells divide symmetrically, producing identical daughter cells. Division orientation is crucial for maintaining tissue architecture. Within an epithelial layer, cells divide along the plane of the tissue to maintain the layer's integrity and allow expansion. In multi-layered epithelia, like the epidermis, cell divisions occur perpendicular to the tissue plane. Loss of spindle control can disrupt the layered architecture During the morphogenesis of epithelial tubes, divisions happen along the tube's length, ensuring that growth in length surpasses growth in circumference. Asymmetric Stem Cell Divisions (ACD) ACDs generate daughter cells with distinct fates. Two primary mechanisms drive ACD: o Intrinsic Mechanisms: Fate determinants are asymmetrically segregated into the two daughter cells. o Extrinsic Mechanisms: The two daughter cells are placed in different microenvironments that influence their fates. In both mechanisms, the orientation of the mitotic spindle is crucial for determining cell fate. Sister Chromatid Cohesion and Separation After replication, sister chromatids are held together by cohesin, a ring-shaped protein complex.  The cohesin complex is composed of Smc1a, SMC3 (both ATPases), and Rad21, forming a tripartite ring structure. Separase, an endopeptidase, cleaves cohesin during the metaphase-to-anaphase transition, allowing sister chromatid separation. Regulation of Separase: o Bound to an inhibitor called securin. o Inhibited by phosphorylation by Kinases The anaphase-promoting complex (APC) triggers the ubiquitin-dependent degradation of cyclin B and securin by the 26S proteasome, leading to separase activation. Cohesin at centromeres is more protected and is removed last, explaining the V-shape of separating chromosomes. Cytokinesis Cytokinesis is the division of the cytoplasm and organelles after nuclear division (mitosis). In animal cells, the cytoplasm pinches in, forming a cleavage furrow. In plant cells, a cell plate forms between the daughter nuclei. Reasons for Cell Division Cells divide for several reasons: 1. Growth: Organisms grow by increasing cell number, not by increasing the size of individual cells. 2. Tissue Repair: Cell division replaces damaged or dead cells. 3. Maintenance of Volume-to-Surface Ratio: As a cell grows, its volume increases faster than its surface area. If a cell becomes too large, it faces difficulties in nutrient uptake and waste removal. Cell division maintains an optimal volume-to- surface ratio. Lecture 6 DNA Damage: A Constant Threat Causes of DNA Damage: DNA damage arises from various sources, categorized as: o Exogenous Damage: External factors like UV radiation and chemical mutagens. o Endogenous Damage: Internal factors like reactive oxygen species (ROS) produced during cellular metabolism.  Types of DNA Damage: DNA damage can manifest in various forms, including: o Small-Scale Mutations: ▪ Point mutations: affect single base or short sequence of bases within a gene. ▪ Substitutions: One base replaced by another ▪ Deletions/insertions ▪ Frameshift: Insertions or deletions changing the reading frame o Large-Scale Mutations: Alterations at the chromosomal level ▪ Duplications: Extra copies of chromosome segments ▪ Deletions: Loss of chromosome segments ▪ Inversions: segment of a chromosome is removed and replaced within the chromosome in reverse order ▪ Translocations: segments of two chromosomes are exchanged o Double-Strand DNA Breaks (DSBs): Severe damage where both strands of the DNA molecule are broken. Consequences of DNA Damage: o Mutations: Permanent changes in the DNA sequence that can alter gene function, leading to various diseases. ▪ Mutations affecting homologous recombination: mutations in Brca1 and Brca2 genes are a cause of hereditary breast and ovarian cancer. o Cell Cycle Arrest: DNA damage can trigger cell cycle checkpoints, halting cell division to allow time for repair. o Senescence: A state of irreversible cell cycle arrest, preventing further cell division. o Apoptosis: Programmed cell death, eliminating cells with irreparable DNA damage. Mutation Rates and Their Significance Low Mutation Rates in Humans: Limitations on Protein Complexity: A higher mutation frequency would drastically increase the probability of critical genes suffering damaging mutations Elevated Mutation Rate in Mitochondria  The Crucial Role of DNA Repair DNA as the Only Repaired Macromolecule Maintaining Genomic Integrity: Precise DNA replication coupled with efficient DNA repair mechanisms are vital for preserving the integrity of the genome. Genetic Investment in Repair: A significant portion of the coding capacity of most genomes is devoted to DNA repair. Link to Diseases: Inadequate DNA repair is linked to diseases, including cancer. Sources of DNA Damage: Endogenous and Exogenous Factors Endogenous Damage: Spontaneous processes within the cell contribute to DNA damage. Examples include: o Depurination: The cleavage of N-glycosidic bonds, releasing A or G bases from DNA, creating apurinic (AP) sites. o Deamination: The removal of an amino group (NH2), most commonly converting cytosine to uracil. Can also occur in DNA. o Reactive Oxygen Species (ROS): Highly reactive molecules like superoxide radicals (H2O2), hydrogen peroxide, and hydroxyl radicals generated during cellular metabolism can directly oxidize DNA bases (add OH). Exogenous Damage: External factors contribute significantly to DNA damage. Examples include: o UV Light: Primarily induces the formation of thymine dimers, where adjacent thymine bases become covalently linked, distorting the DNA structure and hindering replication and transcription. o Chemical Mutagens: Diverse chemical agents can modify DNA bases, leading to various types of damage, including alkylation, cross-linking, and base substitutions. Oxidative DNA Damage and Aging Mitochondrial Respiration as a Major Source: source of ROS G very Susceptible to Oxidation: 8-OHdG Mutagenic Potential of 8-OHdG: causing G:C → T:A mutations. 8-OHdG can mispair with A instead of C. Age-Related Increase in Oxidative Damage Methylation and Replication Stress S-Adenosylmethionine (SAM): SAM serves as the primary methyl donor for various biological methylation reactions, including the methylation of DNA, RNA, proteins, and phospholipids.  DNA Methylation: DNA methyltransferases utilize SAM to methylate cytosines in DNA, primarily at CpG dinucleotides. This process adds a methyl group to the 5th carbon position of the cytosine ring, forming 5-methylcytosine (5-mC). Replication Stress: Replication stress arises when the replication fork, the site of DNA synthesis, encounters obstacles or disruptions. This can lead to fork stalling, collapse, or the generation of aberrant fork structures. o Causes of Replication Stress: ▪ Replication fork barriers ▪ Defects in replication forks: nucleotide imbalance or deletion mutations of the replisome components ▪ Dysregulated Ori firing: defects in licensing, dormant ori can be activated upon replication stress to rescue stalled replication forks and genomic regions deprived of ‘back up’ dormant ori experience higher replicative stress o Various factors can trigger replication stress, including DNA lesions, secondary structures formed by DNA, collisions between the replication machinery and transcription complexes (R-loops), nucleotide imbalance or depletion, and mutations in replisome components. o Oncogene Activation and Oxidative Stress Fork Stalling and Repair: major contributor to genomic instability Fork Stalling and Checkpoint Activation: When the DNA polymerase stalls at a damaged site, the helicase, responsible for unwinding the DNA, may continue to unwind, generating stretches of single-stranded DNA (ssDNA). This ssDNA, bound by Replication Protein A (RPA), activates the replication stress response, leading to the recruitment of ATR kinase, a protein kinase that promotes fork stabilization and blocks cell cycle progression until replication is complete. Fork Rescue and Potential Outcomes: Various mechanisms can rescue stalled forks, allowing replication to continue. However, if the fork collapse occurs, it results in a double-strand DNA break (DSB), a severe form of DNA damage. o Homologous Recombination (HR) Repair: HR repair can accurately repair DSBs by using a homologous DNA template. o Non-Homologous End Joining (NHEJ) Repair: NHEJ repair, an error- prone mechanism, can rejoin broken DNA ends but may introduce mutations or genomic instability. o Cell Death: If the DSB remains unrepaired, it can trigger cell death. DNA Repair Pathways and the Double Helix ssDNA Repair: 1. Direct Reversal: Directly reversing the damage without removing or replacing nucleotides. 2. Mismatch Repair (MMR): Correcting mismatched base pairs and small insertions/deletions introduced during DNA replication. 3. Excision Repair: Removing damaged or incorrect bases by excising a stretch of DNA containing the lesion. This includes: ▪ Base Excision Repair (BER): Removes a single damaged base. ▪ Nucleotide Excision Repair (NER): Removes a larger patch of DNA surrounding the damage. ▪ Ribonucleotide Excision repair (RER) 4. Tolerance Mechanisms: lesion bypass (TLS). dsDNA Repair: 5. Recombinational Repair: ▪ Homologous Recombination (HR) ▪ And Non Homologous End Joining (NHEJ) The Double Helix and Repair: The double-stranded nature of the DNA molecule is crucial for repair. When one strand is damaged, the complementary strand serves as a template to guide the repair process. Direct Repair: Photoreactivation Photoreactivation: A direct repair mechanism where the damage is reversed without removing or replacing nucleotides. Mechanism of Photoreactivation: 1. Photolyase Binding: The enzyme photolyase binds to UV-induced pyrimidine dimers (e.g., T-T dimers) in the DNA. 2. Activation by Light: The bound photolyase absorbs a photon of light, becoming activated. 3. Dimer Cleavage: The activated photolyase breaks the covalent bond between the pyrimidine bases, restoring the original DNA structure. 4. Enzyme Release: The photolyase is released from the DNA. Photolyase: found in various species but not in placental mammals. High Fidelity of DNA Replication: Proofreading and Mismatch Repair High Fidelity DNA Polymerases Exonucleolytic Proofreading: Most DNA polymerases have a separate catalytic site or domain with 3ʹ→5ʹ exonuclease activity. This proofreading function allows the polymerase to remove incorrectly incorporated nucleotides at the 3ʹ end of the growing DNA strand, reducing the error rate.  Mismatch Repair (MMR) Excision Repair Target of Corrects mismatched base pairs Removes damaged or incorrect bases caused repair and small insertions/deletions by chemical modifications or UV radiation that occur during DNA replication Timing Immediately after DNA replication Any time when DNA damage is detected Amount Removes and replaces a single - Base excision repair removes a single of DNA mismatched base damaged base. removed - Nucleotide excision repair removes a larger patch of DNA (~30 nucleotides) surrounding the damage Mismatch Repair (MMR): o corrects mismatched base pairs and small insertions or deletions that escape proofreading during DNA replication. o Takes advantage of ds nature of DNA o Damage is excised o Correct DNA sequence is restored by DNA pol that uses undamaged strand as template o Remaining break is sealed by DNA ligase Mismatch Repair in E. coli Three Steps of MMR: recognition, excision, and gap-filling DNA synthesis. MutS/MutL: better recognition Recognition by MutS: The MutS protein recognizes and binds to mismatched base pairs or small insertion/deletion loops in the DNA. o ATP-Dependent Conformational Change: Upon binding to the mismatch, MutS undergoes an ATP-dependent conformational change, forming a sliding clamp that can move along the DNA. MutS recruits MutL to the DNA, forming a complex that can scan the DNA for a specific signal to identify the newly synthesized strand. The MutS-MutL complex slides along the DNA, searching for a hemi-methylated GATC site, which serves as a marker of the newly synthesized strand. When the MutS-MutL complex reaches a hemi-methylated GATC site, it recruits MutH, an endonuclease, and activates its activity. MutH then nicks the unmethylated strand at the GATC site, providing an entry point for the excision machinery. A DNA exonuclease and a helicase, recruited to the nick and remove the portion of the newly synthesized strand containing the mismatch. DNA polymerase then fills in the resulting gap, and DNA ligase seals the nick. Mismatch Repair in prokaryotes: new strand is unmethylated Mismatch Repair in Eukaryotes utilize the asymmetry of DNA replication and specific protein interactions to identify the newly synthesized strand. Lagging Strand: The lagging strand, presence of nicks (single-strand breaks) between Okazaki fragments marks this strand as newly synthesized. Leading Strand: The mechanism for strand discrimination on the leading strand is less clear but likely involves several factors: Asymmetric PCNA Loading: PCNA (proliferating cell nuclear antigen), a protein that acts as a sliding clamp for DNA polymerases, is loaded onto the DNA in an asymmetric manner during replication. DNA pol [ε, δ, α] randomly incorporate rNTP incised by RNaseH2 -> creates nicks Nicks discriminate which strand to repair nascent DNA strand Incorporation of Ribonucleotides: DNA polymerases may occasionally incorporate ribonucleotides (rNTPs) into the DNA strand. These rNTPs are later removed by the enzyme RNase H2, leaving behind nicks that can serve as strand discrimination signals. Steps of Mismatch Repair in Eukaryotes: 1. Recognition: MutSα (ATPase sites), a eukaryotic homolog of MutS, recognizes and binds to mismatched base pairs or small insertion/deletion loops in the DNA. 2. Cutting: PCNA, loaded onto the DNA during replication, activates the endonuclease activity of MutLα, leading to the nicking of the newly synthesized strand near the mismatch. 3. Mismatch Removal: An exonuclease removes a stretch of DNA containing the mismatch, creating a gap in the newly synthesized strand. 4. DNA Synthesis and Ligation: DNA polymerase fills in the gap using the template strand as a guide, and DNA ligase seals the nick, completing the repair process. Base Excision Repair (BER) 1. Base Removal: A DNA N-glycosylase, specific for a particular type of damaged base, recognizes and removes the damaged base from the DNA, creating an apurinic/apyrimidinic (AP) site, also known as an abasic site. 2. Backbone Nicking: AP endonuclease recognizes the AP site and cleaves the phosphodiester backbone on the 5' side of the missing base, generating a 3'-OH terminus. 3. Gap Filling and Ligation: DNA polymerase extends the 3'-OH terminus, using the undamaged strand as a template, to fill in the gap. DNA ligase then seals the remaining nick, completing the repair. DNA Glycosylases and Base Flipping: Cells possess a variety of DNA glycosylases, each specific for a particular type of damaged base, ensuring comprehensive repair and removal of: o Deaminated Cs o Deaminated As o Alkylated or oxidized bases o Bases with opened rings; DNA glycosylases employ a "flipping-out" mechanism, where they rotate the base out of the DNA helix to probe for damage. This allows the enzyme to access and remove the damaged base. BER Pathways: BER in Bacteria BER in eukaryotes Glycosylase Glycosylase excises a damaged excises a damaged base; base; AP endonuclease AP endonuclease nicks the backbone nicks the backbone at AP site. at AP site. Exonuclease Long patch repair: DNA pol extends the DNA creates a gap strand from the 3′ terminus, displacing the ss DNA; Pol I synthesizes a Flap endonuclease cleaves away ssDNA flap → new strand. ligation. Ligase seals the Short patch repair: only one nucleotide is gap. inserted prior to ligation. Why the U in RNA was replaced in DNA by T (= 5-methyl U). ❑ Spontaneous deamination of C converts it to U. ❑ If DNA contained U, the repair system would not be able to distinguish a deaminated C from a naturally occurring U. Nucleotide Excision Repair (NER) NER is a Flexible repair pathway that deals with bulky DNA lesions that distort the DNA double helix. These lesions can be caused by: covalent reaction of DNA bases with large hydrocarbons (various carcinogens) Pyrimidine dimers (T-T, T-C, and C-C) caused by sunlight. Any damage that distorts the DNA molecule. NER in Prokaryotes: - large multienzyme complex [Uvr A, B, C] scans the DNA for a structural distortion, NOT for a specific base change. - Once a lesion is found, [Uvr A, B, C] cleaves the phosphodiester backbone of the abnormal strand on both sides of the distortion - DNA helicase peels away the s-s oligonucleotide with the lesion. - The large gap is repaired by DNA pol and DNA ligase - Summary: o Damage recognition: Binding of a UvrA2B at the damaged site o Double incision of the damaged strand several nucleotides away from the damaged site, on both the 5’ and 3’ sides o Helicase “peel away” damaged oligonucleotide from between the two nicks o Filling in of the resulting gap by a DNA pol o Ligation - UvrC – major player in NER o Dual Endonuclease activity: ▪ Cleaves the phosphodiester bond DNA on both sides of damage o DNA damage removal: ▪ Excising a 12-nucleotide fragment containing the DNA lesion. o Complex formation: ▪ Binds to UvrB after UvrA dissociates, forming the UvrBC complex at the damage site. o Structural features: ▪ Several domains, including RNase H domain in a 'closed' inactive state o RNase H domain activation: ▪ Through interaction with UvrB and damaged DNA Ribonucleotide Excision Repair (RER) [rNTP] concentrations are much higher than [dNTP] concentrations in cells rNTPs compete with dNTPs for incorporation by DNA polymerases. Step 1: incision on the 5′ side of the rNTP by RNase H2: recognize and cleave a rNTP in a DNA Step 2: Strand displacement synthesis by DNA Pol generates a flap processed by an exonuclease to release the ribonucleotide- containing segment of DNA. Step 3: The resulting nick is sealed by DNA ligase. NER in Eukaryotes: NER in eukaryotes follows a similar pathway but involves a more complex set of proteins. Transcription-Coupled NER (TC-NER): Cells prioritize the repair of actively transcribed genes through TC-NER. This mechanism ensures that the transcribed strand of an active gene is repaired faster than non-transcribed regions. RNA polymerase stalling at a DNA lesion → use of coupling proteins → directs NER to these sites. Translesion Synthesis (TLS) TLS is a damage tolerance mechanism that allows DNA replication to proceed past DNA lesions that block replicative polymerases. A different risky strategy: o High fidelity replicative DNA pol stall when they encounter damaged DNA o Less accurate, backup polymerases (translesion polymerases) are called to action 7 translesion polymerases of different “profession” in humans: o A) Recognize a specific type of DNA damage, add nucleotide to restore the initial sequence. o B) Make “good guesses” if the template was extensively damaged. Translesion Polymerases: Specialized DNA polymerases are recruited to the stalled replication fork. bypass the lesion by incorporating nucleotides opposite the damaged site. Lack exonucleolytic proofreading activity Many are much less discriminating in choosing which nucleotide to incorporate  Each translesion polymerases is given a chance to add few nucleotides before the highly accurate replicative polymerase resumes DNA synthesis. responsible for most of the base-substitution and single-nucleotide deletion mutations in genomes ❑ Produce mutations when copying damaged DNA should be: a) tightly regulated; b) allowed to act only at sites of DNA damage PCNA Ubiquitination and TLS: PCNA ubiquitination plays a crucial role in regulating TLS. When DNA damage is encountered, PCNA is modified by the addition of ubiquitin, a small protein. This ubiquitination serves as a signal to recruit translesion polymerases to the stalled fork. Consequences of TLS: While TLS allows replication to continue, it can introduce mutations due to the error-prone nature of translesion polymerases. However, TLS is essential for cell survival in cases of extensive DNA damage. Lecture 7 (start with nice summary of DNA damage repair for ssDNA) Double-Strand Break Repair DSBs are particularly hazardous forms of DNA damage, leading to chromosome breakdown if left unrepaired. Translocations can be detected by FISH. Exogenous Causes of DSBs Several external factors can induce DSBs in DNA: Ionizing Radiation: This type of radiation interacts with water molecules, causing radiolysis, which breaks down water into reactive oxygen species (ROS). UV Radiation Oxidizing Agents: These agents, often present in the environment or generated as byproducts of cellular metabolism, can attack DNA and cause various types of damage, including DSBs. Topoisomerase Inhibitors: Topoisomerases create transient breaks in DNA to relieve torsional stress. Endogenous Causes of DSBs Transcription-related Conflicts: The cellular machinery responsible for transcribing DNA into RNA can sometimes clash with the machinery replicating DNA. These conflicts can lead to stalled replication forks and subsequent DSB formation.  Meiotic Recombination: programmed DSBs are intentionally created. These breaks are crucial for exchanging genetic material between homologous chromosomes, leading to genetic diversity in offspring. Replication Stress: Stalled replication forks, often caused by DNA damage or obstacles in the DNA template, can break, especially when encountering single- strand nicks or gaps in the parental DNA ahead of the replication fork. Detecting DSBs: The Comet Assay The Comet Assay is a sensitive technique used to detect DSBs in cells. This method involves embedding lysed cells in agarose and subjecting them to electrophoresis. Damaged DNA, which is more fragmented, migrates farther in the electric field, creating a characteristic "comet" shape, with the "tail" of the comet representing the broken DNA fragments. The length and intensity of the comet tail correlate with the extent of DNA damage. Repairing DSBs: Two Major Pathways Non-Homologous End Joining (NHEJ): NHEJ involves a series of steps, including recognition of the broken ends by the Ku heterodimer (Ku70/Ku80), recruitment of other repair factors like DNA-PKcs, processing of the DNA ends, and finally, ligation of the ends by DNA ligase IV. While NHEJ is crucial for maintaining genome stability, its error-prone nature can introduce mutations, such as insertions or deletions, at the site of the break. Homologous Recombination (HR): It involves a complex series of steps, including DNA end resection, strand invasion, D-loop formation, DNA synthesis, and resolution of Holliday junctions. HR ensures accurate repair by using a template, minimizing the introduction of errors. Non-Homologous End Joining Homologous Recombination (NHEJ) (HR) Error-prone mechanism Precise, high-fidelity repair mechanism Repair DSBs with non-cohesive or Uses homologous DNA sequence blunt DNA ends as a template – (sister chromatid) Operates throughout cell cycle Requires a significant stretch of homology (dozens to hundreds bp) Preference for G1/ early S/ G2 Mainly active late S and G2 phases The choice between NHEJ and HR depends on several factors, including the cell cycle phase, the availability of a homologous template, and the structure of the DSB ends: S phase: presence of homologous template (sister chromatid) makes HR the predominant pathway Consequences of DSB Repair Consequences of NHEJ Mutations: error-prone nature, can lead to mutations at the site of the break. The accumulation of these mutations over time contributes to cellular dysfunction and aging - "scars" representing sites of inaccurate NHEJ repair. Chromosomal Rearrangements: The lack of a homology can result in the joining of DNA ends from different chromosomes, leading to chromosomal rearrangements. These rearrangements can create chromosomes with two centromeres or chromosomes lacking centromeres, leading to genomic instability. Consequences of HR Loss of Heterozygosity (LOH): While HR is generally a high-fidelity repair mechanism, it can occasionally lead to LOH. This occurs when the repair machinery uses the homologous chromosome from the other parent instead of the sister chromatid as a template. Major Players in DSB Repair - Depend on kinases Major Players in NHEJ DNA-PKcs (DNA-dependent Protein Kinase catalytic subunit): a serine/threonine kinase that plays a central role in NHEJ to recognize and process ds DNA breaks. It forms a complex with the Ku heterodimer at the site of the DSB, becoming activated upon binding. The activated DNA-PKcs then phosphorylates other repair factors, including Artemis, initiating end processing and promoting NHEJ. The Ku Heterodimer: The Ku heterodimer, composed of Ku70 and Ku80 subunits, is the first responder to DSBs. It rapidly binds to the broken DNA ends, protecting them from degradation and recruiting DNA-PKcs to the site. MRN Complex (MRE11-RAD50-NBS1): involved in both NHEJ and HR. It binds to DSBs, tethers the broken ends, and participates in end processing. The MRE11 subunit possesses both endonuclease and 3′-to-5′ exonuclease activities, allowing it to trim DNA ends and remove damaged nucleotides. Artemis: Artemis is a nuclease that is activated by DNA-PKcs phosphorylation. It works in conjunction with the MRN complex to process the DNA ends at the DSB site, removing damaged or mismatched nucleotides and preparing the ends for ligation. DNA Ligase IV (Lig4): enzyme responsible for sealing the processed DNA ends during NHEJ. Mechanism of NHEJ Repair Process 1. Recognition and Binding: Ku70/Ku80 recognizes the broken DNA ends and recruits DNA-PKcs to the site, forming the DNA-PK holoenzyme. Ku proteins have an affinity for each other, bringing the two broken DNA ends into proximity. 2. Activation: DNA-PK interaction with the DNA termini triggers autophosphorylation, activating DNA-PKcs. 3. Recruitment of Repair Factors: Additional repair factors, including the MRN complex, Artemis, and Lig4, are recruited to the site of the DSB. 4. End Processing: DNA-PK phosphorylates and activates Artemis. Artemis, along with the MRN complex, processes the DNA ends by removing damaged or mismatched nucleotides and trimming the ends. 5. Filling Gaps: If there are gaps between the processed ends, DNA polymerase fills them in. 6. Ligation: Lig4 seals the processed DNA ends, completing the repair of the DSB. DNA-PKcs, besides its role in NHEJ, also acts as a signal transducer in the DNA damage response (DDR). Upon activation, it transmits signals downstream, activating various cellular processes in response to DNA damage: Cell Cycle Checkpoints: DNA-PKcs activation triggers cell cycle checkpoints, leading to a temporary halt in cell cycle progression (p53). This pause allows time for DNA repair before the cell proceeds to the next phase of the cycle. Senescence: In cases of extensive or irreparable damage, DNA- PKcs can induce cellular senescence, a state of permanent cell cycle arrest. Apoptosis: If the damage is too severe, DNA-PKcs can trigger apoptosis. Beneficial Roles of NHEJ V(D)J Recombination: help produce immune cells / antibodies. Class Switch Recombination (CSR): This process allows B cells to switch from producing one class of antibody (e.g., IgM) to another (e.g., IgG, IgA). HR: An Accurate Pathway for DSB Repair HR is a highly accurate pathway for repairing DSBs. This pathway utilizes a homologous DNA sequence as a template for repair. HR is crucial for maintaining genomic integrity and plays a particularly important role in meiosis:  Meiotic Recombination: During meiosis, programmed DSBs are created to facilitate the exchange of genetic material between homologous chromosomes. These breaks are repaired by HR, leading to the formation of hybrid chromosomes with novel combinations of genes. This process is essential for generating genetic diversity in offspring. Crossing Over: HR during meiosis results in the formation of a four-way structure at the point of strand exchange, called a Holliday junction. It leads to crossing over, the exchange of genetic material between homologous chromosomes. Regulation of HR In meiosis, HR is favored between homologous chromosomes, promoting the exchange of genetic material and generating diversity. However, if a similar event occurs in mitosis, it can lead to detrimental consequences, such as LOH. In somatic cells, HR preferentially utilizes the sister chromatid as a template for repair, minimizing the risk of LOH. Loss of Heterozygosity (LOH) occurs when the repair machinery uses the homologous chromosome from the other parent instead of the sister chromatid as a template. If the homologous chromosome carries a deleterious mutation, the repair process can result in the loss of the functional allele. Cell Cycle Coordination of HR The first step in HR is resection of the broken ends, generating single- stranded DNA (ssDNA) overhangs. The nucleases involved in resection are most active in the S and G2 phases when a sister chromatid is available as a template. In somatic cells, the proximity of the two daughter chromatids favors their use for repair, making HR preferentially utilize sister chromatids over homologous chromosomes as template donors. HR initiation is most efficient in S-phase for several reasons: o The presence of a sister chromatid provides a readily available homologous template. o The long ssDNA overhangs generated by end resection inhibit the NHEJ pathway. Long ssDNA prevents the binding of the Ku heterodimer, shifting the balance towards HR. "Strand Dance" and Strand Invasion The process of HR involves a remarkable "strand dance," where the damaged strand searches for its complementary strand to use as a template for repair. 1. The ends of the broken DNA are recognized and processed by the MRN complex, which digests the 5' ends to produce overhanging 3' ssDNA ends. 2. Strand invasion: One of the 3' ssDNA ends from the damaged DNA invades the template duplex, searching for homologous sequences – creates a D-loop. This process is facilitated by Rad51, a protein that forms a filament on the ssDNA and promotes strand exchange. DNA End Resection generating 3' ssDNA overhangs that are essential for strand invasion. Initiation of DNA end resection: The MRN complex generates a short ssDNA at the DSB ends. Extension of DNA end resection: Once the initial ssDNA is generated, downstream exonucleases, such as exonuclease 1 (EXO1), extend the 3'- ssDNA. This extension is crucial for RPA (Replication Protein A) and RAD51 loading, which are essential for strand invasion. Regulation of DNA end resection: Uncontrolled end resection can lead to genomic instability. Therefore, multiple mechanisms exist to terminate end resection when sufficient ssDNA length is generated for HR repair. Role of BRCA1 and BRCA2: BRCA1 and BRCA2 are tumor suppressor proteins that play crucial roles in HR. BRCA1 promotes end resection by interacting with the MRN complex. BRCA2 helps in the loading of RAD51 onto the ssDNA. Strand Exchange and D-loop Formation Role of Rad51: eukaryotic homolog of the bacterial RecA protein, is a crucial player in strand exchange. It forms a filament on the ssDNA coated with RPA, a ssDNA binding protein that protects the ssDNA from degradation. Rad51 then displaces RPA from the ssDNA, facilitating strand invasion into the homologous duplex. This displacement promotes homology searching and strand pairing. D-loop Formation: The invading strand, guided by Rad51, forms a D-loop by displacing one of the strands of the homologous duplex. DNA polymerase then extends the invading strand, using the homologous strand as a template. Double Holliday Junction Formation and Resolution In some cases, HR proceeds through the formation of a double Holliday junction (dHJ), a four-way DNA junction that connects the damaged and template DNA molecules.  dHJ Formation: The invading strand, after DNA synthesis, can be captured by the other end of the DSB, leading to the formation of a dHJ. dHJ Resolution: Holliday junction resolvases are specialized enzymes that cleave dHJs, separating the entangled DNA molecules. The resolution of dHJs can lead to either crossover or non-crossover products. BRCA1 and BRCA2: Guardians of Genome Integrity BRCA1 and BRCA2 are tumor suppressor proteins that play crucial roles in HR and maintaining genomic stability. BRCA1: BRCA1 promotes end resection BRCA2: BRCA2 facilitates the loading of RAD51 onto ssDNA, promoting strand invasion and HR. Minor damage = cell cycle paused → damage repaired Medium damage = cell cycle stopped → damage repaired – but if stopped for too long = senescent cell Strong damage = cell cycle aborted → cell death DNA Damage Response (DDR) Cell Cycle Checkpoints: The DDR activates cell cycle checkpoints, halting cell cycle progression to allow time for DNA repair. DNA Repair: The DDR recruits and activates DNA repair machinery to the sites of damage. Apoptosis: If the damage is too extensive or irreparable, the DDR can trigger apoptosis Senescence: In some cases, the DDR can induce senescence, a state of permanent cell cycle arrest, preventing the proliferation of cells with damaged DNA. p53: Guardian of the Genome The tumor suppressor protein p53 plays a central role in the DDR. p53 Regulation: In undamaged cells, p53 is kept at low levels by Mdm2, a ubiquitin ligase that targets p53 for degradation by proteasomes. p53 Activation: DNA damage, including ATM DSBs, DNA-Pk and ATR replicative stress, stabilizes p53, leading to its accumulation and activation. p53 Functions: Activated p53 acts as a transcription factor, regulating the expression of genes involved in cell cycle arrest, DNA repair, and apoptosis. If damage is not too bad: p21 pauses cell cycle briefly If damage moderate: p21 causes senescence If damage TOO BAD: BAX causes apoptosis p53 → transcription of p21 and BAX  o Cell Cycle Arrest: p53 induces the expression of p21, a cyclin-dependent kinase inhibitor that halts cell cycle progression, allowing time for DNA repair. o Apoptosis: If the damage is irreparable, p53 promotes the expression of pro-apoptotic genes like BAX, leading to cell death. DNA Damage Sensing Kinases Specialized kinases play crucial roles in sensing DNA damage and activating the DDR: ATM (Ataxia Telangiectasia Mutated): ATM is activated by DSBs. It phosphorylates a variety of substrates, including p53, checkpoint kinases, and DNA repair proteins, promoting DNA repair and cell cycle arrest. ATR (ATM and Rad3-related): ATR is activated by ss DNA breaks, including stalled replication forks. It phosphorylates substrates involved in stabilizing stalled forks, preventing their collapse into DSBs, and promoting HR. DNA-PK: DNA-PK is activated by DSBs. It phosphorylates substrates involved in NHEJ, promoting this repair pathway. γH2A.X: A Platform for Repair Factors H2A.X is a histone variant of H2A that plays a crucial role in the DDR, particularly in response to DSBs. Phosphorylation of H2A.X: forming γH2A.X. γH2A.X: platform for the recruitment of DNA repair factors and other DDR proteins to the sites of DSBs. The accumulation of these proteins at DSBs forms visible foci that are used as markers to detect DSBs and assess the efficiency of DNA repair. Canonical HJ resolvases. Enriched in (+) charged amino acids, consistent with their ability to bind DNA with high affinity. Dimeric enzymes with twin active sites to catalyze two coordinated incisions.  Symmetrical resolution: 2 coordinated and symmetrically related nicks at, or very near, the branch point. Lecture 8 Genomes and genes Gene: a specific DNA sequence coding for a functional product: a protein or a non-coding RNA molecule. Common view: Much of noncoding DNA is dispensable junk, retained like old papers because it is easier to retain everything. Repetitive elements: ~ 50% of the genome. Up to 80% of the human genome is transcribed into RNA. Most of the repetitive elements are transposons. Transposons = transposable elements (TEs) = jumping genes Segments of DNA that can move [transpose] from one location to another within a genome. In humans: ~45-65% of the genome. Types of transposons based on their mechanism of movement: Move via an RNA intermediate: ~ 40% Move directly as DNA "cut and paste": >2% Examples of transposons in humans: LINE1/ L1 and Alu Transposons’ activity varies between individuals and populations. Most human transposons are silent and immobile. Estimates focus on germline insertions that can be inherited. Retroviruses vs Retro-TE Retroviruses and LTR- Retro-TE share a common structural organization. The main difference: LTR- RetroTE lack a functional envelope (Env) gene. LTRs Flank the internal coding region of TE, defining its boundaries. Contain enhancer and promoter driving transcription of TE. Contain both the start and termination sites for transcription. Essential for the integration of TE into new genomic locations. In humans, non-LTR RetroT is a more active class of transposons capable of jumping Non-LTR RetroT ORFs: ORF0 (if present): o located in the antisense direction within the 5'UTR of LINE1; o transcribed from an antisense promoter. ORF1 (when present) o RNA binding peptide  o Nucleic acid chaperone ORF2 (always present) o Reverse transcriptase (RT) domain: essential for their mobilization. o Endonuclease domain L1 The most abundant non-LTR of the human genome. Over 100 genetic diseases have been attributed to germline L1-mediated retro-transposition. Alu ORF0 (if present): located in the antisense direction within the 5' UTR of LINE1; transcribed from an antisense promoter. How non-LTR retrotransposons (like LINE-1) integrate into the genome 1. Transcription: L-1 is transcribed from the promoter in 5’UTR by RNA pol II, producing a bicistronic mRNA: ORF1 and ORF2. 2. mRNA Processing: L-1 mRNA is 5'-capped and 3'-polyadenylated and exported from the nucleus to the cytoplasm. 3. ORF1/ORF2 RNP Translation: (ORF1) → ORF1p: RNA-binding nucleic acid chaperone that binds to L-1 RNA; (ORF2) → ORF2p: endo-nuclease and RT; it also binds to L-1 RNA 4. Ribonucleoprotein Complex (RNP): ORF1p + ORF2p + L-1 RNA 5. Retro-transposition Process o Ribonucleoprotein (RNP)s enter the nucleus at M phase when the nuclear envelope breaks down. o In the nucleus: ORF2p initiates target-primed reverse transcription, where L-1 RNA serves as a template for synthesizing complementary DNA. o EN (endonuclease)→ s-s nick in its consensus recognition site: 5′- TTTTA-3′; L1-RNA anneals with polyA-tail to TTTT-motif, serving as a primer for RT. How RNA