Molecular Biology I: Nucleic Acid Metabolism Lecture 5 2024S1 PDF

MOLECULAR BIOLOGY I: NUCLEIC ACID METABOLISM SC/BIOL 3110, 2024 S1 Lecture 05: DNA replication Telomeres and telomerase Molecular Biology Techniques 1 Recap of last lecture: Leading and lagging strand synthesis at the replication bubble: Detection of Okazaki fragments supported the semi-discontin...

MOLECULAR BIOLOGY I: NUCLEIC ACID METABOLISM SC/BIOL 3110, 2024 S1 Lecture 05: DNA replication Telomeres and telomerase Molecular Biology Techniques 1 Recap of last lecture: Leading and lagging strand synthesis at the replication bubble: Detection of Okazaki fragments supported the semi-discontinuous synthesis model. 2 Recap of last lecture: 3 main steps in DNA replication: Initiation, elongation, termination: Tus tus 36,000 Da Termination of replication 3 Recap of last lecture: Initiation of DNA replication in E. coli: DnaC released in order for helicase to start Note: DnaA only binds to negatively supercoiled and fully methylated (on both strands) OriC à Helps melting/denaturing of dsDNA at AT-rich region à Ensures that OriC is only active (licenced) once per round of DNA replication 4 Recap of last lecture: Initiation / elongation steps of replication : DNA synthesis cannot initiate on its own – must have RNA primer base paired to template DNA first Therefore, start of DNA synthesis requires synthesis of RNA primers by DnaG, and elongation by DNA polymerases Synthesis occurs in 5’ to 3’ direction and requires 5’ tri-phosphate to provide energy to form phosphoester bond 5 Recap of last lecture: Initiation / elongation steps of replication : DNA polymerase responsible for elongation of newly synthesized DNA strand All polymerases have similar structural features and functions Mg2+ ions (on palm of polymerase) help orient incoming nucleotides Fingers play a role in checking correct base pairing – i.e. fidelity Palm of replicative polymerase has ideal shape and size (~ 22 Å x 30 Å) for B-form DNA 6 Recap of last lecture: E. coli DNA polymerase III holoenzyme: = main replicative polymerase = multi-subunit holoenzyme with 4 subassemblies “Clamp loader” 7 Recap of last lecture: E. Coli DNA pol III at replication fork: DNA pol III + b-rings = highly processive Primase = distributive Note: the b-rings and clamp loader are missing in this figure 8 Recap of last lecture: E. Coli DNA replication: The last few steps of lagging strand synthesis: 1. Removal of RNA primer 2. Filling in the gap 3. Ligation of Okazaki fragments 9 Recap of last lecture: E. Coli DNA replication termination: DNA replication forks disassemble when they encounter non-permissive Tus-bound Ter sites. Permissive vs non-permissive Ter/Tus sites are relative to direction of replication fork. Tus Helicase is stopped by Tus protein if it is coming from non-permissive side. Passage of replication fork from permissive side displaces Tus Stoppage of replication fork at non-permissive Ter/Tus 10 Recap of last lecture: Fidelity of E. coli replication: Polymerase selectivity 1. Correct base pairing 2. Size selection of correct nucleotide at catalytic site of polymerase 3. Quality control by O-helix (part of Fingers) before phospho-ester linkage 3’ to 5’ exonuclease proofreading Mismatch repair mechanism E. coli error rate: ~ 1 in 109 nts added Eukaryotes: ~ 1 in 1010 nts added 11 Recap of last lecture: E. coli methyl-directed mismatch repair: MutH can scan for methylated GATC (methylated at A) on either side of the mismatch and then nick the opposite strand. 12 Recap of last lecture: E. coli methyl-directed mismatch repair: Another example of mismatch repair assay: à Incubate mismatch plasmid with cell lysates, recover DNA and cut with REs Sal IR Sal I site = GTCGAC This strand must be methylated in order for normal pathway to repair mismatch to generate Sal I site Sal IS 13 DNA replication and related processes: Eukaryotic DNA replication: Replication of eukaryotic cells is more complex and less well characterized. Basic steps of DNA synthesis and properties of DNA polymerases are mostly conserved between prokaryotes and eukaryotes. However, eukaryotic cells à genomes are much larger, and are chromatinized. Also, chromosomes are linear, with lots of origins of replication. DNA replication is strictly confined to S phase of cell cycle. Rate of replication is much slower (~ 75 nts/s) compared to prokaryotes(~ 1000 nts/s) à likely due to chromatin. Most chromosomal replicons do not have termination region like that of E. coli. 14 DNA replication and related processes: Eukaryotic DNA replication: Eukaryotic cells have many origins of replication. In yeast, origins are well defined and also called ARS (autonomously replicating sequences). In higher eukaryotes, origins are not well defined and difficult to identify. In higher eukaryotes, it is clear that not all potential origins of replication fire at the same time during replication. Regulation of origin firing is complex and actively investigated by research labs still. 15 DNA replication and related processes: Eukaryotic DNA replication: 1. The origin recognition complex (ORC) binds to DNA and provides a site on the chromosome where additional replication factors can associate. 2. Pre-replicative complex formation involves the association of Mcm2-7 complex with DNA at ORC. 3. Mcm2-7 proteins provide helicase activity for DNA synthesis and loading of these proteins confers competence on the origin to fire in S phase. 4. Onset of DNA synthesis requires the action of two protein kinases (cyclin dependent kinase (CDK) and Cdc7), which trigger the association of additional proteins with the origin. During the process of initiation, DNA polymerases are also recruited and DNA synthesis starts. 5. During replication, Mcm2-7 proteins move away from the origin and further assembly of pre-replicative complexes is blocked. This ensures that origins can only fire a single time 16 per cell cycle. Early G1 S G2 Simplified summary of steps in eukaryotic DNA replication DNA replication and related processes: Eukaryotic DNA replication: Once origin of replication fires, events/proteins at the replication forks are similar to that of E. coli. Pol a = primase + another DNA polymerase activity. Single strand binding protein is called RPA (Replication protein A). PCNA = b ring of DNA pol III. Eukaryotic clamp loader is called RFC (Replication factor C), and is highly similar to the prokaryotic clamp loader complex. Recent findings showed that leading strand is synthesized by Pol e whereas lagging strand switches between Pol a and Pol d. 17 DNA replication and related processes: Eukaryotic DNA replication: Main eukaryotic polymerases (there are additional ones): For base excision repair 18 DNA replication and related processes: Eukaryotic DNA replication: Cycles of switching between DNA Pol a and Pol d on the lagging strand: 1. Pol a synthesizes RNA primer (10 – 12 nts) and then ~30 nts of DNA 2. RFC displaces Pol a and recruits PCNA with Pol d 3. PCNA clamps Pol d on DNA 4. Pol d elongates Okazaki fragment synthesis 19 DNA replication and related processes: Eukaryotic DNA replication: DNA polymerase switching and processing of an Okazaki fragment on the lagging strand. A.As the DNA helicase promotes unwinding at the replication fork, DNA pol e with RFC and PCNA synthesizes DNA on the leading strand. DNA pol α initiates synthesis on the lagging strand by generating an RNA primer (red segment) followed by a short segment of DNA. Then, RFC and PCNA load a second DNA polymerase (d) to continue synthesis of the Okazaki fragment. A.As DNA pol d approaches the downstream Okazaki fragment, cleavage by RNase H1 removes the initiator RNA primer leaving a single 5′-ribonucleotide. Then, FEN1/RTH1 removes the last 5′-ribonucleotide. The resulting nick is sealed by DNA ligase. 20 DNA replication and related processes: Comparison of prokaryotic and eukaryotic DNA replication proteins: Note that PCNA is made up of homo-trimer whereas b-clamp is made up of dimer 21 DNA replication and related processes: Eukaryotic DNA replication: Termination of DNA replication in eukaryotes – not well defined. End when replication forks run into each other? 22 DNA replication and related processes: DNA replication in the context of chromatin: Eukaryotic DNA replication is even more complicated given presence of chromatin and nucleosomes. Need to disassemble chromatin ahead of the replication fork, and re-assemble nucleosomes post DNA replicaiton. PCNA directly binds to chromatin remodeling complexes such as CAF-1. 23 DNA replication and related processes: Fidelity of DNA replication in eukaryotes: Basically same as in prokaryotes, however, larger selection of polymerases with different error rates. High fidelity polymerases Low fidelity polymerase for by passing DNA lesions 24 DNA replication and related processes: Eukaryotic mismatch repair: Again, similar to prokaryotic system except not methyl-directed. Initiated by nick on one strand à = repaired strand 25 End replication problem, telomeres and telomerase Eukaryotic DNA replication: End replication problem: Chromosomes of eukaryotic cells are linear – so the DNA replication machinery cannot replicate the very end of the lagging strand (Watson, Olovnikov, 1970s). 27 Eukaryotic DNA replication: How to solve the end replication problem? Most eukaryotic chromosomes end in direct repeat sequences called telomeres. All telomeres have G-rich strand that ends as single strand overhang. In human, the repeating 6-mer = TTAGGG (aka T2AG3). 28 Eukaryotic DNA replication: How to solve the end replication problem? All chromosomes have same telomeric sequences, but different chromosomes may have different lengths of telomeres. Can use telomere-specific probes in FISH to visualize telomeres, and also to measure telomeric lengths 29 Molecular Biology techniques: Fluorescence In Situ Hybridization (FISH): “In situ” means in their natural positions within a chromosome. FISH can be applied to visualize specific genes on chromosomes, or detection of localized RNAs within the cell 30 Eukaryotic DNA replication: How to solve the end replication problem? In somatic cells of humans, telomeres shorten with age. So in somatic cells, there is no mechanism to overcome the end replication problem. Gradual loss of telomeres also known as telomere erosion. 31 Eukaryotic DNA replication: Telomere length shortening: Telomeric length can be measured by Southern blot or by fluorescence microscopy analyses. Genomic Southern blot to measure the lengths of the telomeric TRFs (terminal restriction fragments) from human cells Quantitative FISH analysis of metaphase chromosomes 32 Eukaryotic DNA replication: Telomere length shortening: Telomeric length shortening correlates with chronological ageing. Declining average telomere length in DNA extracted from the peripheral blood of 60 individuals between 0 to 85 yrs of age 33 Eukaryotic DNA replication: Telomeric ends have to be protected: The 3’ overhang of telomeres need to be protected, otherwise will be recognized as damaged DNA by repair mechanisms. Overhang can fold into T-loop/D-loop or in G-quartet structure, and also protected by a variety of telomere-binding proteins. T-loop D-loop G-quartet EM picture of a T-loop isolated from human tissue culture cells 34 Eukaryotic DNA replication: Telomeric ends have to be protected: A large variety of proteins bind to the telomeres of different organisms. Some of these proteins bind to the single strand overhang of the telomere (e.g. POT1), but others bind to the double-strand portion of the telomere. Some are even interspersed between nucleosomes. In mammals, these protective proteins (TRF1, TRF2, POT1, TPP1, RAP1 and TIN2) form a complex called Shelterin. In mammals, the double-strand part of telomeric DNA is also organized in tightly packed nucleosomes with shorter repeat lengths (compared to bulk nucleosomes), and they also exhibit hallmarks of heterochromatin. 35 Eukaryotic DNA replication: How to solve the end replication problem? Chromosomes with critically short telomeres tend to form end-to-end fusions to protect the ends à genomic instability! telomere erosion e.g. loss of p53 or Rb senescence 36 Eukaryotic DNA replication: How to solve the end replication problem? Telomere shortening is linked to replicative aging. Normal cells have finite lifespan à Hayflick limit – most cells stop growing and senescence. Small population continue to grow till they hit crisis. 37 Eukaryotic DNA replication: How to solve the end replication problem? However, some cells such as germ cells or stem cells have stable telomere lengths. Also, cancer cells can bypass crisis and grow indefinitely. 38 Eukaryotic DNA replication: How to solve the end replication problem? In cells such as stem cells or germ cells, they have special enzyme called telomerase to re-generate telomeres. Greider and Blackburn (mid 80s) set out to biochemically purify telomerase in Tetrahymena à turns out this organism has unique property that made it an excellent source of telomerase. Tetrahymena is the perfect organism for biochemical purification of telomerase activity because during its vegetative development, they generate a functional nucleus (macronucleus) from the genomic nucleus (micronucleus). This process not only involves amplification of the DNA content (each gene is amplified ~ 40X), but also the addition of numerous telomeres to the ends of each amplified chromosome. 39 Eukaryotic DNA replication: How to solve the end replication problem? Greider and Blackburn deviced an in vitro activity assay to measure telomerase activity to help them purify components of telomerase. Found that telomerase has two distinct components – activity sensitive to both RNase and protease, therefore: An RNA component A protein component no template needed! They cloned the gene responsible for the RNA component, but identification of the protein component took another 10 years (done by other groups). In vitro reaction ran on sequencing gel showed incorporation of radioactive dGTP on the extended oligo primer 40 Eukaryotic DNA replication: How to solve the end replication problem? Greider and Blackburn discovered that telomerase is made up of protein catalytic enzyme (known as TERT) and an RNA component (TER) à discovery led to Nobel prize in 2009. The RNA component has partial complementarity to the G-rich strand overhang à telomerase utilizes RNA component as template for elongation of G-rich strand. Telomerase is a type of reverse transcriptase (copy RNA to make DNA). 41 Eukaryotic DNA replication: How to solve the end replication problem? Blackburn and colleagues proved that the RNA component of telomerase serve as template for telomeres in Tetrahymena by mutating the gene that transcribe the RNA component (TER RNA). Did similar experiment in human cancer cells and found that this caused them to die 42 Eukaryotic DNA replication: To solve the end replication problem: Telomerase specifically extends the G-rich strand of telomeres, and C-rich strand is filled in by the regular DNA replication machinery. An unknown mechanism maintains a single-stranded overhang on the G-rich strand at the end of chromosomes. 43 Eukaryotic DNA replication: Too much telomerase is not a good thing: In humans, the RNA component is transcribed in all cells, but the protein component (TERT) is only expressed in germ/stem cells. Introduction of the hTERT gene into mortal cells not only increased their telomere lengths, but also allowed them to proliferate indefinitely (“immortalized” cell line). Average lengths of telomeres from chromosomes isolated from TERT- or TERT+ cells Problem – cancer cells also often activate TERT expression to allow them to grow indefinitely. 44 Eukaryotic DNA replication: Is re-activating telomerase a good thing? Mice engineered to lack telomerase age prematurely. Re-introduction of telomerase into these mice can reverse the premature aging phenotype. 45 Eukaryotic DNA replication: Telomerase and aging: Myth linking red wine drinking and longevity Resveratrol thought to be key molecule in red wine responsible for this 46 Eukaryotic DNA replication: Resveratrol – enhancer of telomerase? Some scientists claimed that resveratrol activates an enzyme called SirT1 known to regulate telomerase activity and telomere length; however, later found to be due to an artifact of the biochemical assay à still very controversial These and other myths have spawned a huge but bogus resveratrol- (and telomerase-) based industry. 47 Molecular Biology methods and techniques Methods in Molecular Biology – useful enzymes: Phosphatases and nucleases: Phosphatases: hydrolyze ester bond to remove phosphate group Nucleases: hydrolyze ester bond in phosphodiester linkage between nucleotides degrade nucleic acids endonucleases hydrolyze internal bonds exonucleases chew from the ends also RNases that specifically target RNAs RNase H targets RNA strand of RNA/DNA hybrid The target of a phosphatase (a) and a nuclease (b). An endonuclease (c) and an exonuclease (d) 49 Methods in Molecular Biology – useful enzymes: Restriction endonucleases: AKA restriction enzymes, derived from bacteria and archea Highly DNA sequence-specific endonucleases that serve to protect bacteria from foreign DNA Convention for naming REs: 50 Methods in Molecular Biology – useful enzymes: Restriction endonucleases: The most common REs often belong to the Type II category Type II enzymes recognize and cleave the same DNA sequences, whereas Type I and Type III enzymes recognize specific sequences, but cut at a distal site (Type III à 20, 30 bp from the recognition site, or Type I à up to 1 Kb away) For Type II enzymes, the recognition/cleavage sites average between 4 – 8 bp, and typically are palindromic in nature Also generally require Mg2+ as co-factor Note the difference between 5’ overhangs and 3’ overhangs 51 Methods in Molecular Biology – useful enzymes: Restriction endonucleases: Enzymes that have the same recognition sequence are called isoschizomers e.g. Hpa II (C/CGG) and MspI (C/CGG) Enzymes that have the same recognition sequence cut differently are called neoschizomers e.g. Aat II (GACGT/C) and Zra I (GAC/GTC) Some enzymes are sensitive to the methylation of DNA whereas others are not. e.g. HpaII cannot cleave CCGG when the second C is methylated, whereas MspI will cleave sequence regardless of methylation status Some enzymes have different recognition sequences but leave “compatible” overhangs à very useful for cloning different pieces of DNA together 52 Methods in Molecular Biology: Cloning: To clone, means to make identical copies, so cloning refers to making copies of, or amplifying a DNA fragment or gene of interest Paul Berg (in his 1972 paper) was the first to combine genes from different organisms, which resulted in the formation of recombinant DNA Used restriction enzymes to cut open DNA from SV40 (monkey virus) and lambda bacteriophage (bacterial virus) and engineered a “cut-and-splice” method by joining these pieces of DNA through the sticky ends generated by the REs and by the use of DNA ligase 53 Methods in Molecular Biology: Restriction mapping: Which model is correct? Method used to map an unknown segment of DNA by digesting with restriction enzymes and identifying the location of the RE cleavage sites 54 Methods in Molecular Biology: Restriction mapping: Method used to map an unknown segment of DNA by digesting with restriction enzymes and identifying the location of the RE cleavage sites 55 Methods in Molecular Biology: Reagents important for cloning: 1. Enzymes: a. Restriction endonucleases b. DNA ligase c. Phosphatase/kinase 56 Methods in Molecular Biology: Reagents important for cloning: Note what are compatible vs non-compatible ends for ligation: 57 Methods in Molecular Biology: Reagents important for cloning: 2. Vectors: Common vectors used in cloning: There are also expression vectors that are specifically engineered to express (transcribe and translate) gene of interest in the appropriate host organisms Bacterial Artificial Chromosome Yeast Artificial Chromosome 58 Methods in Molecular Biology: Reagents important for cloning: 2. Vectors: An expression vector would contain a bacterial or mammalian cell promoter for transcribing gene of interest cloned into the MCS For a gene to be translated into protein, must contain ATG start codon for translation initiation A typical plasmid for cloning 59 Methods in Molecular Biology: Reagents important for cloning: 3. Antibiotics: Used in combination with the specific antibiotic resistant gene present on the vector of choice Used for selection à i.e. to inhibit growth of E. coli that do not contain plasmid-ofinterest E.g. Ampicillin, Kanamycin b-lactam ring Amp = b-lactam antibiotic with an amino group side chain attached to the penicillin structure Penicillin derivative that inhibits bacterial cell wall synthesis by inhibiting peptidoglycan cross linking Mode of resistance: the b-lactamase (bla) gene cleaves the b-lactam ring of Amp 60 Methods in Molecular Biology: Typical scheme for cloning (i.e vector + insert): Transform plasmids into competent bacteria (e.g. DH5a strain of E. coli) by heat shock (e.g. 42oC for 2 min) Plate onto agar plates containing appropriate antibiotics 61