DNA Replication Chapter 9 PDF

1 Chapter 9: DNA Replication General features of DNA replication - Double helical model for DNA: - Two strands are complementary - Each strand can serve as a template for synthesizing its complementary partner - DNA replication follows a semiconservative mod...

1 Chapter 9: DNA Replication General features of DNA replication - Double helical model for DNA: - Two strands are complementary - Each strand can serve as a template for synthesizing its complementary partner - DNA replication follows a semiconservative model - Replication is half-discontinuous, producing short fragments that are later joined together - Requires RNA primers to initiate replication - Usually occurs in a bidirectional manner THE CHEMISTRY OF DNA SYNTHESIS DNA synthesis requires deoxynucleoside triphosphates and a primer: template junction - dNTP: (dATP, dCTP, dATP and dGTP). It has three phosphate attached to the 5’hydroxyl of the 2’-deoxyribose. - Template: provide the ssDNA that directs the addition of each complementary deoxynucleotides. - The primer is complementary to, but shorter than the template. It must have a free 3’-OH adjacent to the ssDNA region of the template. - Essential substrates for DNA synthesis: dNTPs and primer: template junction (a combination of dsDNA and ssDNA regions) DNA is synthesized by extending the 3’ end of the primer - Initiation of DNA synthesis: - The 3’-OH group of the primer attacks the α-phosphate of the incoming dNTP - This extends the 3’ end of the primer by one nucleotide - Releases one molecule of pyrophosphate - Pyrophosphate: quickly hydrolyzed into phosphate molecules by pyrophosphatase - DNA strand orientation: the template strand is oriented opposite to the growing DNA strand THE MECHANISM OF DNA POLYMERASE DNA polymerases use a single active site to catalyze DNA synthesis  2 - DNA polymerase function: monitors the ability of incoming nucleotides to form correct base pairs (A:T or G:C) rather than identifying the exact nucleotide - Correct base pairing: - Only when a proper base pair is formed are the 3’-OH of the primer and the α-phosphate of the incoming dNTP positioned optimally for catalysis - This mechanism serves as an example of kinetic proofreading Steric constraints preventing DNA polymerase from using rNTP precursors - rNTP vs. dNTP incorporation: - Despite rNTPs being present at 10 times higher concentration in cells, they are incorporated over 1000 times less frequently than dNTPs - Discrimination mechanism: - Steric exclusion prevents rNTPs from fitting into the DNA polymerase active site - The nucleotide-binding pocket cannot accommodate the 2’-OH group of rNTPs - Role of discriminator amino acids: - These amino acids make van der Waals contacts with the sugar ring - Altering these amino acids to those with smaller side chains (e.g., changing glutamate to alanine) reduces the polymerase’s ability to discriminate between dNTPs and rNTPs Chemotherapeutic reagents that target DNA replication - Nucleotides that do not fully meet DNA polymerase requirements can inhibit DNA synthesis by terminating elongation - These inhibitors are used as anti-cancer or anti-viral drugs: - 5-fluorouracil: Pyrimidine analog - 6-mercaptopurine: Purine analog - Cytosine arabinoside: Deoxycytidine analog; terminates elongation due to the structural difference between deoxyribose and arabinose - Cisplatin: Forms intrastrand DNA cross-links - Bis-chloroethylnitrosourea: Alkylating agent that creates intrastrand DNA cross-links - Azidothymidine (AZT): Thymidine analog that inhibits reverse transcriptase; used as an anti-viral agent - Acyclovir: Guanine analog lacking the ribose group and 3’-OH, preventing further nucleotide addition - Cisplatin: the platinum atom binds covalently to the N7 of purines), forming interstrand and intrastrand crosslinking.  3 Measuring DNA Polymerase activity (incorporation assay) - 1. [α-32]dATP - 2. Fluorescently labeled thymidine triphosphate analog DNA Polymerase resembles a hand that gri[s the primer:template junction - Palm domain: - Composed of a β-sheet - Contains the primary elements of the DNA polymerase catalytic site - Binds two divalent metal ions (usually Mg²⁺ or Zn²⁺) - Monitors the base-pairing of recently added nucleotides through extensive hydrogen-bond contacts with the minor groove of the new DNA - Thumb domain: - Not directly involved in catalysis - Interacts with the DNA that has just been synthesized Two metal ions bound to the DNA Polymerase catalyze nucleotide addition - Role of metal ions in DNA synthesis: - Metal ion A: - Interacts with the 3’-OH group of the primer - Reduces the association between oxygen and hydrogen, creating a nucleophilic 3’-O⁻ - Metal ion B: - Binds to the triphosphate of the incoming dNTP - Neutralizes the negative charge of the triphosphate - Stabilizes the pyrophosphate product after catalysis - T7 DNA Polymerase: a specialized DNA polymerase used for high-fidelity replication in bacteriophage T7  4 DNA Polymerase “grips” the template and the incoming nucleotide when a correct base pair is made - Fingers domain: - Composed of α-helices - Contains residues that bind to the incoming dNTP - Upon correct base pairing between the dNTP and the template, the fingers enclose the dNTP, stimulating catalysis - Key interactions: - Tyrosine (Tyr): Forms stacking interactions with the base of the dNTP - Lysine (Lys) and Arginine (Arg): Associate with the triphosphate of the dNTP to stabilize it The Path of the template DNA through the DNA Polymerase - The finger domain associates with the template region. - This interaction causes a nearly 90° turn in the phosphodiester backbone. - The turn occurs between the first and second base of the template. - As a result, only the first template base is exposed to the catalytic site. DNA Polymerases are processive enzymes - Catalysis by DNA polymerase is rapid, adding up to 1000 nucleotides per second to a primer strand. - This speed is due to the processive nature of DNA polymerase. - Processivity is defined as the average number of nucleotides added per binding event at the primer:template junction. - Processivity is enhanced by the sliding of DNA polymerase along the DNA template. - Once bound, DNA polymerase interacts tightly with the double-stranded DNA in a sequence-independent manner. - Key interactions include: - Electrostatic interactions between the thumb domain and the phosphate backbone. - Interactions between the palm domain and the minor groove of DNA. - When a nucleotide is added, the DNA partially releases from the polymerase, breaking hydrogen bonds in the palm domain while maintaining electrostatic interactions with the thumb. - The DNA rapidly rebinds to the polymerase, shifted by one base pair, using the same sequence-nonspecific mechanism.  5 Summary of DNA Polymerase Domains - Palm domain: - contain the primary elements of the catalytic site - Monitor the base pairing of the most recently added nucleotides: by making extensive hydrogen-bond contact with the base pairs in the minor groove of the newly synthesized DNA - Finger domain: - bind and enclose the incoming dNTP - Associate with the template region, leading to 90 degree turn of the phosphodiester backbone between the first and second bases in the template - Thumb domain: (not intimately involved in catalysis) - maintain the correct position of the primer and the active site - Maintain a strong association between the DNA Polymerase and phosphate backbone of the substrate DNA, good for DNA polymerase processivity Exonucleases proofread newly synthesized DNA - DNA Replication Error Rate: 1 in 10^10. - DNA Polymerase Error Rate: 1 in 10^5. - Proofreading Exonuclease: - Originally identified in the same peptide as DNA polymerase. - Degrades DNA starting from the 3’ end. - Exonuclease: degrades DNA from an end of the DNA strand. - Endonuclease: cuts within a DNA strand. - Incorrect Nucleotide Incorporation: leads to reduced DNA synthesis rate due to improper positioning of the 3’-OH group. - Presence of Mismatched 3’ End: - Last 3-4 nucleotides of the primer become single-stranded. - Increased affinity for the exonuclease active site occurs. - The mismatched nucleotide is removed from the primer once bound at the active site. - Post-Correction Process: - After the mismatched nucleotide is removed, a properly base-paired primer:template junction is reformed. - DNA polymerization resumes. - Role of 3’-Exonuclease in DNA Polymerase: - The exonuclease function is part of the same peptide as DNA polymerase, termed the “delete key.” - This increases the accuracy of DNA synthesis. - Error Rate with Proofreading Exonuclease: - With proofreading, the error rate of DNA synthesis decreases to 1 in 10^7. THE REPLICATION FORK Both strand of DNA are synthesized together at the replication fork  6 - DNA Replication: both strands of the DNA duplex are replicated, requiring the separation of the two strands to create two template DNAs. - Replication Fork: defined as the junction between replicated and unreplicated DNA. - Semidiscontinuous Replication: occurs in E. coli and other organisms. - Leading Strand: replicated continuously in the direction of the replicating fork movement. - Lagging Strand: replicated discontinuously as short segments (Okazaki fragments). - Length of Okazaki fragments: 1-2 kb in bacteria//100-400 bp in eukaryotes. - Synthesized in the opposite direction of the replication fork. - Directionality: both strands are replicated in the 5’ to 3’ direction. The initiation of a new strand of DNA requires an RNA primer DNA Polymerase I - DNA Polymerase I is specialized for the removal of the RNA primers that are used to initiate DNA synthesis. - Pol I is not highly processive, adding only 20-100 nucleotides per binding event. - DNA polymerase I (pol I) is a versatile enzyme with 3 distinct activities - DNA polymerase - 3’-->5’ exonuclease - 5’-->3’ exonuclease: remove the RNA-DNA linkage that is resistant to RNase H. - Mild proteolytic treatment results in 2 polypeptides - Klenow fragment (the large domain) - Smaller fragment Klenow Fragment - Contains both: Polymerase and 3’-->5” exonuclease activity, which serves as proofreading - If DNA polymerase I (Pol I) adds an incorrect nucleotide, it will not base pair properly. - Pol I pauses, and the exonuclease activity removes the mispaired nucleotide. - This allows replication to continue correctly. - The proofreading function increases the fidelity of DNA replication. Eukaryotic DNA Polymerases - There are more than 15 DNA polymerases.  7 - Three essential DNA polymerases: - DNA Pol δ: Specializes in synthesizing the lagging strand. - DNA Pol ε: Specializes in synthesizing the leading strand. - DNA Pol α/primase - Composed of two subunits: DNA Pol α and a two-subunit primase. - Functions in initiating new DNA strand synthesis. - Most of the other DNA polymerases are involved in DNA repair. DNA Polymerase switching during eukaryotic DNA replication - Because of its low processivity DNA Pol α/primase is rapidly replaced by DNA Pol δ and DNA Pol ε Sliding clamps dramatically increase DNA polymerase processivity - 3D structure of sliding DNA clamp: - Composed of multiple identical subunits, forming a doughnut-like shape. - The central hole has a diameter of approximately 34 Å, larger than the 20 Å width of the DNA helix. - This size allows for a thin layer of one or two water molecules between the sliding clamp and the DNA, facilitating smooth movement along the DNA strand. - Interaction with DNA Polymerase: - The sliding DNA clamp interacts with the part of the DNA polymerase nearest to the newly synthesized DNA as it emerges from the enzyme. - Without the sliding clamp, DNA polymerase tends to dissociate and diffuse away from the template DNA after synthesizing an average of 20-100 base pairs. - The sliding clamp helps prevent DNA polymerase from diffusing away, keeping it attached to the DNA and enhancing processivity. - Once DNA polymerase completes synthesis of the template, the absence of a primer:template junction triggers a change in the polymerase. - This change causes DNA polymerase to release from the sliding clamp.  8 3D structure of sliding clamp from different organism - Sliding clamp structures: - E. coli: Composed of two copies of the β protein. - T4 phage: Consists of a trimer of gp45 protein. - Eukaryotes: Formed by a trimer of PCNA (Proliferating Cell Nuclear Antigen). - Sliding clamps exhibit conserved sixfold symmetry. - After DNA polymerase releases, sliding clamps remain attached to the replicated DNA. - Functions of PCNA in eukaryotes: - Recruits enzymes for chromatin assembly. - Interacts with proteins involved in Okazaki fragment repair. Sliding clamps are opened and placed on DNA by clamp loaders - Clamp loader: - A 5-subunit protein complex. - To open the sliding clamp, the clamp loader must bind ATP. - Mechanism: - ATP binding enables the clamp loader to bind and open the sliding clamp at one of the subunit interfaces. - This allows the clamp-loader complex to bind DNA, specifically at primer:template junctions. - The double-stranded DNA (dsDNA) is positioned in the “hole” of the sliding clamp. - ATP hydrolysis triggers the release of the clamp loader. - Interaction with DNA polymerase: - Sliding clamp loaders and DNA polymerase have overlapping binding sites on the sliding clamp, preventing them from interacting simultaneously. - Role in replication initiation: - The sliding clamp is part of the holoenzyme but is not part of DNA polymerase during replication initiation. - This contributes to the low processivity of DNA polymerase α (or DNA Pol I). - Multiple functions of the sliding clamp: - It interacts with various proteins, including DNA Pol IV, for DNA repair. DNA SYNTHESIS AT THE REPLICATION FORK Composition of the DNA Pol III holoenzyme - At the replication fork, the simultaneous synthesis of the leading and lagging strands minimizes the presence of single-stranded DNA (ssDNA), which is more challenging to repair if damaged. - This coordinated strategy involves multiple DNA polymerases working together at the replication fork. - DNA Pol III Holoenzyme structure: - Contains three copies of the DNA Pol III core enzymes.  9 - Includes one sliding clamp loader composed of five subunits. - The sliding clamp loader has three τ proteins, each interacting with one DNA Pol III core enzyme.l Trombone Model of DNA replication in E.Coli - Lagging strand synthesis: - Two DNA Pol III core enzymes work alternately to initiate synthesis of new Okazaki fragments on the lagging strand. - One DNA Pol III core enzyme continuously replicates the leading strand. - Role of DNA helicase: - DNA helicase moves along the lagging strand template in the 5’ to 3’ direction. - DNA Pol III holoenzyme interacts with DNA helicase via the τ protein. - ssDNA protection: - Single-stranded DNA (ssDNA) regions are coated with single-strand binding proteins (SSBs) to stabilize them. - RNA primer synthesis and clamp loading: - DNA primase associates periodically with DNA helicase to synthesize new RNA primers on the lagging-strand template. - The sliding clamp loader assembles a sliding DNA clamp at the primer:template junction. - Okazaki fragment synthesis: - The unengaged second lagging-strand DNA polymerase recognizes the loaded sliding clamp and begins synthesizing a new Okazaki fragment. - The first lagging-strand DNA polymerase releases from the sliding clamp upon reaching the end of an Okazaki fragment and is then ready to initiate the next fragment. - “Trombone” model: - Describes the dynamic change in the size of the ssDNA loop formed between DNA polymerase and DNA helicase on the lagging-strand template, resembling a trombone slide as the loop lengthens and shortens. Binding of the DNA helicase to DNA Pol III holoenzyme stimulates the rate of DNA strand separation - τ subunit interactions at the replication fork: - The τ subunit of the sliding clamp loader interacts with both DNA helicase and DNA polymerase. - Coordination of DNA unwinding and replication: - When DNA helicase and DNA polymerase are associated, DNA helicase unwinds the DNA at the same rate as DNA polymerase replicates it. - If DNA helicase is not associated with the DNA Pol III holoenzyme, the unwinding rate of DNA helicase slows by 10-fold.  10 - Under these conditions, DNA polymerases replicate faster than DNA helicase can separate the DNA strands, allowing DNA Pol III holoenzyme to catch up and re-establish the replisome. - DNA helicase and primase interaction: - DNA helicase interacts with DNA primase, significantly stimulating primase activity. INITIATION OF DNA REPLICATION The replicon model of replication initiation - General initiation of DNA synthesis: - DNA synthesis usually begins at internal regions of the DNA. - Replicon model (1963, Francois Jacob et al): - Describes how replication initiation is controlled in bacteria. - Replicon: A specific DNA region required for the initiation of DNA replication. - Components of the replicon model: - Replicator: - A cis-acting DNA sequence sufficient to direct the initiation of replication. - Often includes a stretch of AT-rich DNA. - The origin of replication is a part of the replicator where DNA unwinds and synthesis begins, though the origin may be only a fraction of the entire replicator. - Initiator: - A protein that specifically recognizes a DNA element within the replicator and activates replication initiation. - Contains a core AAA+ ATP-binding motif, regulated through ATP binding and hydrolysis. - The initiator protein is the only DNA-binding protein that recognizes DNA sequences to initiate replication. - AAA/AAA+ ATPases: refers to ATPases associated with diverse cellular activities, sharing a conserved domain of around 230 amino acid residues. Replicator sequences include initiator-binding sites and easily unwound DNA - Structure of replicators: - Initiator binding site (9-mer (green) in Ecoli) - Easily unwound DNA (13-mer (blue), AT-rich in E coli) - (red): the site of initial DNA synthesis Genetic identification of replicators  11 Identify the location of origins of replication in cells - 2D Agarose Electrophoresis: A technique used to analyze DNA fragments. - Bubble Shape: DNA fragments with an origin of replication form a distinct “bubble” shape. - Y-Shaped Structure: DNA fragments lacking an origin of replication exhibit various “Y-shaped” forms. - The investigator must already know the approximate location of the potential origin of replication to use this method effectively. - Shape Similarity: The Y-shaped molecule from an almost fully replicated fragment resembles a linear molecule that is twice the size of the unreplicated fragment (pattern #3). - Migration Speed: - Y-shaped molecules with three equal-length arms migrate the slowest among similar molecules derived from a specific DNA fragment. - Y-shaped molecules with very short replicated arms or large replicated regions migrate similarly to the unreplicated version of the same DNA fragment. - Bubble-to-Arch Transition: This transition is easily detected as a discontinuity in the arc and is highly indicative of the origin of replication (d). BINDING AND UNWINDING: ORIGIN SELECATION AND ACTIVATION BY THE INITIATOR PROTEIN Initiator function - Initiator Function: - Binds to replicator DNA, typically at a specific binding site.  12 - Interacts with additional factors necessary for replication initiation, such as DNA helicase. - Some initiators distort and unwind a region of DNA adjacent to their binding site to help open the DNA duplex. - DnaA (Initiator Protein): - Binds to specific DNA elements known as 9-mer and 13-mer repeats. - Binding to the 13-mer repeats (in the DnaA-ATP state) involves a unique single-strand DNA-binding site within DnaA. - This interaction causes the separation of DNA strands over more than 20 base pairs within the 13-mer repeat region. Eukaryotic initiator - Eukaryotic Initiator: - Comprised of a six-protein complex known as the Origin Recognition Complex (ORC). - Functionality: - Similar to DnaA, ORC binds and hydrolyzes ATP; ATP binding is essential for sequence-specific DNA binding at the origin. - ATP hydrolysis is necessary for ORC to facilitate the loading of eukaryotic DNA helicase onto the replicator DNA. - Differences from DnaA: - Unlike DnaA, ORC binding to the yeast replicator does not cause strand separation of adjacent DNA. - ORC is essential for recruiting all other replication proteins to the replicator, either directly or indirectly. E. Coli DNA replication is regulated by DnaA·ATP and SeqA A. Methylation State Before Replication: GATC sequences throughout the E. coli genome are methylated on both strands (indicated by red hexagons). B. Transition to Hemimethylated State: DNA replication converts these sites to a hemimethylated state. C. SeqA Binding: Hemimethylated DNA is rapidly bound by SeqA protein. D. Inhibition by SeqA: Bound SeqA inhibits the full methylation of the GATC sequences and the binding of OriC by DnaA proteins. E. Methylation by Dam: SeqA infrequently dissociates from the GATC sites, allowing for full methylation by Dam DNA methyltransferase, which adds a methyl group to the adenine within each GATC sequence. F. DnaA Binding for Replication: once the GATC sites are fully methylated, DnaA binds to the 9-mer sequences, initiating a new round of replication from the daughter oriC replicators.  13 Origins of replication reinitiate replication before cell division in rapidly growing cells - Rapid Growth of E. coli: E. coli can divide every 20 minutes under fast growth conditions. - Genome Replication Time: Replicating the E. coli genome takes over 40 minutes. - Reinitiation of Replication: cells may reinitiate replication once or even twice before completing the previous rounds of replication. - Limitation on Replication per Division: despite reinitiation, DNA replication does not occur more than once per round of cell division; it prepares for the next round instead. - Replication Origin: for each round of cell division, there is only one round of replication initiated from oriC. Protein-protein interaction and protein-DNA interactions direct the initiation process A. DnaA Binding: multiple DnaA ATP proteins bind to the repeated 9-mer sequences within the oriC. B. Strand Separation: this binding leads to strand separation within the 13-mer repeats. C. Association of DNA Helicase and Loader: DNA helicase (DnaB) and the helicase loader (DnaC) associate with the DnaA-bound region. D. Helicase Ring Opening: the helicase loader catalyzes the opening of the DNA helicase ring and places the ring around the single-stranded DNA (ssDNA) at the origin. E. Primase Recruitment: the DNA helicases each recruit primase and synthesize RNA primers on each template. F. Clamp Loader Recognition: a. The newly synthesized primers and helicase are recognized by the clamp loader components of the DNA Pol III holoenzyme. b. Sliding clamps are assembled on each RNA primer, initiating leading strand synthesis by one of the three core DNA Pol III enzymes. G. Lagging-Strand Primer Synthesis: a. After each helicase moves approximately 1000 bases, a second primer is synthesized on each lagging-strand template, and a sliding clamp is loaded. b. The resulting primer-template junction is recognized by a second DNA Pol III enzyme, initiating lagging-strand synthesis. H. Initiation of Synthesis: leading-strand and lagging-strand synthesis are now both initiated at each replication fork. Eukaryotic chromosomes are replicated exactly once per cell cycle - Chromosomal Replication Timing: - Chromosomal DNA replicates only once during the S phase of the cell cycle.  14 - Consequences of Incomplete Replication: - Incomplete replication followed by chromosomal segregation can lead to stress on unreplicated DNA as chromosomes are pulled apart, resulting in chromosome breakage. - Multiple Origins of Replication: - Eukaryotic cells possess many origins of replication; however, not all replicators are activated during a single round of cell division. Replicators are inactivated by DNA replication - Initial Activation: replicators 3 and 5 are the first to be activated. - Inactivation of Copies: activation of a parental replicator leads to the inactivation of the corresponding copies on both daughter DNA molecules until the next cell cycle. - DNA Elongation: elongation of replicators 3 and 5 replicates DNA that overlaps with replicators 2 and 4 before they initiate replication (referred to as passive replicators). - Independent Initiation: replicator 1, not reached by an adjacent replicator, is able to initiate replication normally. - Redundancy for Complete Replication: the presence of more replicators than necessary ensures the complete replication of each chromosome, providing redundancy in the replication process. Helicase loading is the first step in the initiation of replication in eukaryotes - Cell Cycle Timing: - Eukaryotic replication initiation occurs at distinct times in the cell cycle: helicase loading happens during the G1 phase, while replicator activation occurs upon entry into the S phase (differing from prokaryotes). - Origin Recognition Complex (ORC) Binding: - The ATP-bound origin recognition complex (ORC) associates with the replicator. - Recruitment in G1 Phase: - During the G1 phase, ORC bound to the origin recruits two helicase loading proteins (ATP-bound cdc6 and Cdt1) along with two copies of the Mcm2-7 helicase. - Dimer Loading and Release: - The assembly of these proteins triggers ATP hydrolysis by cdc6, which facilitates the loading of a head-to-head dimer of the Mcm2-7 complex around the double-stranded origin DNA, leading to the release of cdc6 and Cdt1 from the origin. - Helicase Release and Process Reset: - Subsequent ATP hydrolysis by ORC is necessary for the release of the helicase and resetting the process for future rounds of replication.  15 Activation of loaded helicase leads to assembly of eukaryotic replisome - Activation of Kinases: - As cells enter the S phase, two kinases, CDK (cyclin-dependent kinase) and DDK (DBF4-dependent kinase), are activated. - DDK phosphorylates the loaded Mcm2-7 helicase, while CDK phosphorylates Sld2 and Sld3. - Binding to Dpb1: - Phosphorylated Sld2 and Sld3 bind to Dpb1, facilitating further steps in the replication process. - Formation of CMG Complex: - Together, these proteins enable the binding of helicase-activating proteins (Cdc45 and GINS) to form the CMG complex with Mcm2-7. - Cdc45 and GINS significantly enhance the ATPase and helicase activities of the Mcm2-7 complex. - Recruitment of Leading-Strand Polymerase: - The leading-strand polymerase (Pol ε) is recruited to the helicase at this stage. - After the formation of the CMG complex, Sld2, Sld3, and Dpb11 are released from the origin. - Recruitment of Lagging-Strand Polymerase: - DNA Pol α/primase and Pol δ (for lagging strand synthesis) are recruited only after DNA unwinding has occurred, which is different from the recruitment of Pol ε. Helicase activation alters helicase interactions - Helicase Loading: - Before helicase activation, loaded helicases are in the form of a head-to-head double hexamer that encircles double-stranded DNA. - Helicase Activation: - After helicase activation, the Mcm2-7 proteins in the complex are proposed to encircle single-stranded DNA. - The interaction between the two Mcm2-7 complexes is broken during this activation process. Helicase loading and activation are regulated to allow only a single round of replication during each cell cycle - Replication Stages: - There is an oscillation between two key replication stages: helicase loading and activation. - Helicase Loading Phase: - Occurs during the G1 phase. - No helicase activation is permitted in this phase.  16 - Activation Phase: - Occurs during the S, G2, and M phases. - Helicase loading is inhibited during this phase, but loaded helicases are activated (activation specifically occurs in the S phase). - After the S phase, all loaded Mcm2-7 complexes will be removed from the DNA. Cell cycle regulation of CDK activity controls replication - CDK Function: - CDK (cyclin-dependent kinase) inhibits helicase loading by affecting the functions of ORC, Cdc6, and Cdt1. - CDK Activity in Cell Cycle: - CDK levels are low during the G1 phase. - Entry into the S phase is associated with a rapid increase in CDK activity, which drives helicase activation. Similarity between eukaryotic and prokaryotic DNA replication initiation FINISHING REPLICATION - Circular chromosome: separate topologically linked DNA - Linear chromosome: complete replication at the very ends of the chromosome Type II Topoisomerases are required to separate daughter DNA molecules - Catenane: a general term for two linked circular DNA molecules. - Type II Topoisomerase: enzymes that break double-stranded DNA (dsDNA) molecules and allow a second dsDNA molecule to pass through the break, helping to resolve issues of DNA topology.  17 - Eukaryotic Chromosomes: - Eukaryotic chromosomes are linear in structure, but their large size necessitates intricate folding into loops. - These loops are attached to a protein scaffold, which aids in organization and compaction. - The attachment of DNA to the protein scaffold can lead to similar challenges as those faced by circular chromosomes during replication, such as resolving catenanes and managing topological stress. The end replication problem - Lagging-Strand End Replication Problem: - As the replication machinery on the lagging strand approaches the chromosome end, primase eventually runs out of space to synthesize a new RNA primer. - Even if an end primer is present, once it is removed, a short region of single-stranded DNA (ssDNA) remains unreplicated at the 3’ end of the lagging strand. - Resulting Incomplete Replication: - This incomplete replication leaves a short ssDNA overhang at the 3’ end of the lagging-strand DNA product. - Progressive Shortening Over Rounds: - In the next round of DNA replication, one of the two resulting DNA products will be shorter, as it will lack the portion of DNA that was not fully copied in the previous replication cycle. - This progressive shortening, if unaddressed, leads to gradual telomere shortening over successive cell divisions.u Protein priming as a solution to the end replication problem - Protein Priming Mechanism: - A specific protein binds to both DNA polymerase and the 3’ end of the DNA template. - This protein provides a priming hydroxyl group, often from a tyrosine residue, to initiate DNA synthesis. - Application in DNA Synthesis: - In some viruses, this protein-priming mechanism initiates all DNA synthesis, acting as an alternative to RNA primers. - For longer DNA molecules, such as in bacterial chromosomes, this protein priming may work in conjunction with conventional origin-based replication to complete the process. - This protein-priming mechanism is particularly useful in viral and bacterial systems that lack access to typical primer-based replication methods, allowing them to efficiently replicate their genomes. Replication of the telomeres by telomerase - Telomere Structure:  18 - Telomeres are the ends of eukaryotic chromosomes and are typically composed of head-to-tail repeats of TG-rich DNA sequences. - In humans, the telomeric sequence is 5’-TTAGGG-3’. - Telomerase Function: - Telomerase is an enzyme that extends the telomeres. - It uses an RNA component within the enzyme to anneal to the 3’ end of the single-stranded DNA (ssDNA) region of the telomere. - Through its reverse transcription activity, telomerase synthesizes DNA complementary to its RNA template, extending the DNA strand. - After synthesizing DNA to the end of the RNA template, telomerase displaces the RNA from the newly synthesized DNA, rebinds to the end of the telomere, and repeats the process to further extend the telomere. Telomerase is a novel DNA Polymerase that doesn’t need an exogenous template - RNA Component: - Telomerase RNA (TER): The RNA component of telomerase, typically ranging from 150 to 1300 bases in length. - Contains about 1.5 copies of the sequence complementary to the telomere, which anneals to the 3’ end of the telomere to guide DNA synthesis. - Protein Components: - Telomerase Reverse Transcriptase (TERT): The protein component that acts as a DNA polymerase, using the RNA template (TER) to synthesize DNA. - TERT carries out reverse transcription to extend the telomeric DNA. - Mechanism of Action: - Telomerase must be able to displace its RNA template from the DNA product, allowing multiple rounds of template-directed synthesis. - This function requires an RNA DNA helicase activity within telomerase to continually reset and reposition the RNA template. - Role in Cell Growth and Aging: - Telomerase plays a significant role in regulating cell growth and aging, as described by the “telomere hypothesis.” - The Hayflick limit suggests that telomere shortening limits the number of cell divisions, contributing to cellular aging when telomerase is inactive in most somatic cells. Extension of the 3’ end of the telomere by telomerase solves the problem of end replication - Telomerase Extends the 3’ End: - Telomerase extends the 3’ end of the chromosome by adding telomeric repeats. This produces a longer 3’ overhang of single-stranded DNA (ssDNA). - 5’ End Extension Using Lagging-Strand Machinery:  19 - After telomerase extends the 3’ end, the lagging-strand DNA replication machinery synthesizes complementary DNA on the opposite strand to extend the 5’ end. - Primase adds an RNA primer, allowing DNA polymerase to synthesize the complementary strand toward the 5’ end. - Remaining 3’ Overhang: - Even after the lagging-strand machinery fills in most of the complementary strand, a short 3’ ssDNA overhang remains at the chromosome end. - This 3’ overhang is significant for telomere function, as it plays a role in protecting the chromosome end. - Telomere Maintenance: - Through the combined actions of telomerase and lagging-strand machinery, the telomere is maintained at a sufficient length. - This maintenance protects chromosome ends from progressive shortening and preserves genomic stability. Telomere-binding proteins regulate telomerase activity and telomere length - (a) Yeast (S. cerevisiae) Telomere-Binding Proteins: - Rap1: Binds directly to double-stranded telomere repeat DNA. - Rif1 and Rif2: Bind to Rap1 and play a role in inhibiting telomerase activity. - Cdc13: Binds to single-stranded telomere repeat DNA and is involved in recruiting telomerase. - (b) Human Telomere-Binding Proteins (Shelterin Complex): - TRF1 and TRF2: Bind directly to double-stranded telomere repeat DNA. - Shelterin Complex: A protective protein complex consisting of TRF1, TRF2, Rap1, TIN2, TPP1, and POT1. This complex protects telomeres from DNA repair enzymes that might otherwise treat telomere ends as DNA damage.  20 - POT1: Binds directly to single-stranded telomere repeat DNA and inhibits telomerase activity, which differs from its role in yeast. - In both yeast and human cells, these telomere-binding proteins regulate telomere length and structure, ensuring chromosome end protection and controlling telomerase access to telomeres.\ Telomere length regulated by telomere-binding proteins - Short Telomeres: - When telomeres are short, fewer telomere-binding proteins are bound to the telomere. - This reduced binding leads to weaker inhibition of telomerase, allowing telomerase to access and extend the 3’ end of the telomere. - Elongated Telomeres: - As the telomere is extended and regions become double-stranded through lagging-strand DNA synthesis, additional telomere-binding proteins can associate with the telomere. - The binding of these proteins increases inhibition of telomerase, preventing further elongation. - Negative Feedback Mechanism: - This process serves as a negative feedback loop: as telomeres reach sufficient length, increased binding of telomere-associated proteins inhibits further telomerase activity, maintaining telomere length within a controlled range. - This feedback mechanism ensures that telomeres do not become excessively long, balancing telomerase activity to protect chromosome ends while avoiding unnecessary elongation. Telomere-binding proteins protect chromosome end - DNA Break Recognition Issue: - DNA ends are typically recognized by the cell as double-strand breaks, which triggers DNA repair mechanisms. - The repair machinery usually initiates recombination with other DNA sequences, which would be harmful if it involved telomeres, as it could lead to chromosomal instability. - Telomere Protection Mechanism: - Telomere-Binding Proteins: Specific proteins bound to telomeres help distinguish them from other DNA ends, preventing them from being recognized as breaks. - If these protective proteins are removed, telomeres are recognized as double-strand breaks, and repair machinery targets them as such. - T-Loop Structure: - Under electron microscopy (EM), isolated telomeres form a t-loop structure, where the 3’ single-stranded overhang loops back and invades the double-stranded region of the telomere. - This t-loop configuration “hides” the chromosome end, preventing it from being mistaken for a DNA break.  21 - Role of TRF2: - The TRF2 protein is capable of directing the formation of t-loops with purified telomere DNA. - The t-loop may also contribute to the control of telomere length, as it helps regulate telomerase access and activity. - By forming t-loops and binding specific proteins, telomeres are protected from repair processes that would otherwise be damaging, ensuring chromosome stability and controlled telomere length.  22 HOMEWORK #7, 8, 11, 14 7) - Position "d" is closest to the replication fork on the 3' end, which makes it the correct location for primase to synthesize the next RNA primer. Position "d" is chosen because primase needs to initiate the next Okazaki fragment near the replication fork, where new single-stranded DNA is being exposed as the fork moves forward. 8) a) Replication proceeds in both directions. b) Bottom. The bottom strand acts as the leading-strand template on the right side. DNA polymerase extends the 3' end of the RNA primer bound to this strand, allowing continuous replication to the end of the template. c) Bottom. DNA ligase is essential for forming phosphodiester bonds between Okazaki fragments on the lagging strand during DNA synthesis. On the left side, the bottom strand serves as the lagging strand template, as DNA synthesis occurs discontinuously in this region. 11)  23 a) DNA helicase is responsible for unwinding the double-stranded DNA at the replication fork. It breaks the hydrogen bonds between the complementary bases, creating single-stranded DNA templates that can be used for replication by DNA polymerase. b) As DNA helicase unwinds the DNA, it creates tension and supercoiling ahead of the replication fork. Topoisomerases relieve this strain by creating temporary breaks in the DNA, allowing it to unwind and release the torsional stress. This action helps DNA helicase function more efficiently by preventing the DNA from becoming too tightly coiled and obstructing the replication process. c) In PCR (Polymerase Chain Reaction), DNA helicase is not required because the process relies on heat to denature the DNA. High temperatures break the hydrogen bonds between the DNA strands, separating them into single strands. This eliminates the need for helicase to unwind the DNA, as the thermal cycling provides strand separation. 14) a) The α-phosphate is integrated into the newly synthesized DNA strand via a nucleophilic attack by the 3'-OH group. The β- and γ-phosphates are released as pyrophosphate, which is subsequently hydrolyzed and not incorporated into the extending DNA strand. b) Gel electrophoresis separates molecules based on size. The ³²P-labeled dNTPs are much smaller than the newly synthesized DNA, allowing them to migrate significantly faster than any long DNA strand. c) A negative control example is running the DNA synthesis assay without DNA polymerase. Without new DNA synthesis, the primer-template junction won’t be labeled. By filtering this reaction, containing the primer-template junction and ³²P-labeled dNTPs, over a positively charged membrane, you should observe no radioactivity adhering to the filter. This can be compared with a reaction that includes DNA polymerase. If there’s concern about potential effects from ³²P-labeled dNTPs binding to DNA polymerase, a protease treatment can be applied before filtering.