DNA REPLICATION- Ramya Rani Vootla

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DNA Replication By Ramya Rani Vootla Lecturer in Biotechnology Ramya Rani Vootla

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Chapter 3: DNA Replication Alternative models of DNA replication Semiconservative model: Meselson-Stahl Experiment DNA synthesis and elongation DNA polymerases Origin and initiation of DNA replication Prokaryote/eukaryote models (circular/linear chromosomes) Telomeres (replicating the ends of chromosomes) Ramya Rani Vootla

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Alternative models of DNA replication (Fig 3.1): Ramya Rani Vootla

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Equilibrium density gradient centrifugation (Box 3.1) Ramya Rani Vootla

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1958: Matthew Meselson & Frank Stahl’s Experiment Semiconservative model of DNA replication (Fig. 3.2) Ramya Rani Vootla

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1955: Arthur Kornberg Worked with E. coli . Discovered the mechanisms of DNA synthesis. Four components are required: dNTPs: dATP, dTTP, dGTP, dCTP (deoxyribonucleoside 5’-triphosphates) (sugar-base + 3 phosphates) 2. DNA template DNA polymerase I (formerly the Kornberg enzyme ) (DNA polymerase II & III discovered soon after) 4. Mg 2+ (optimizes DNA polymerase activity) 1959: Arthur Kornberg (Stanford University) & Severo Ochoa (NYU) Ramya Rani Vootla

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Three main features of the DNA synthesis reaction: DNA polymerase I catalyzes formation of phosphodiester bond between 3’-OH of the deoxyribose (on the last nucleotide) and the 5’-phosphate of the dNTP. Energy for this reaction is derived from the release of two of the three phosphates. DNA polymerase I “finds” the correct complementary dNTP at each step in the lengthening process. rate ≤ 800 dNTPs/second low error rate 3. Direction of synthesis is 5’ to 3’ Image credit: Protein Data Bank Ramya Rani Vootla

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DNA elongation (Fig. 3.4b): Ramya Rani Vootla

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DNA elongation (Fig. 3.4a): Ramya Rani Vootla

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Not all polymerases are the same (Table 3.1) Polymerase Polymerization (5’-3’) Exonuclease (3’-5’) Exonuclease (5’-3’) #Copies I Yes Yes Yes 400 II Yes Yes No ? III Yes Yes No 10-20 3’ to 5’ exonuclease activity = ability to remove nucleotides from the 3’ end of the chain Important proofreading ability Without proofreading error rate (mutation rate) is 1 x 10 -6 With proofreading error rate is 1 x 10 -9 (1000-fold decrease) 5’ to 3’ exonuclease activity functions in DNA replication & repair. Ramya Rani Vootla

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Origin of replication (e.g., the prokaryote example): Begins with double-helix denaturing into single-strands thus exposing the bases. Exposes a replication bubble from which replication proceeds in both directions. Fig. 3.9 ~245 bp in E. coli Ramya Rani Vootla

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Initiation of replication, major elements: Segments of single-stranded DNA are called template strands . Gyrase (a type of topoisomerase ) relaxes the supercoiled DNA. Initiator proteins and DNA helicase binds to the DNA at the replication fork and untwist the DNA using energy derived from ATP ( adenosine triphosphate ). (Hydrolysis of ATP causes a shape change in DNA helicase) DNA primase next binds to helicase producing a complex called a primosome (primase is required for synthesis), Primase synthesizes a short RNA primer of 10-12 nucleotides, to which DNA polymerase III adds nucleotides. Polymerase III adds nucleotides 5’ to 3’ on both strands beginning at the RNA primer . The RNA primer is removed and replaced with DNA by polymerase I, and the gap is sealed with DNA ligase . Single-stranded DNA-binding (SSB) proteins (>200) stabilize the single-stranded template DNA during the process. Ramya Rani Vootla

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Model of replication in E. coli (Fig. 3.5) Ramya Rani Vootla

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DNA replication is continuous on the leading strand and semidiscontinuous on the lagging strand: Unwinding of any single DNA replication fork proceeds in one direction. The two DNA strands are of opposite polarity, and DNA polymerases only synthesize DNA 5’ to 3’. Solution: DNA is made in opposite directions on each template. Leading strand synthesized 5’ to 3’ in the direction of the replication fork movement. continuous requires a single RNA primer Lagging strand synthesized 5’ to 3’ in the opposite direction. semidiscontinuous (i.e., not continuous) requires many RNA primers Ramya Rani Vootla

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3 Polymerase III 5’  3’ Leading strand base pairs 5’ 5’ 3’ 3’ Supercoiled DNA relaxed by gyrase & unwound by helicase + proteins : Helicase + Initiator Proteins ATP SSB Proteins RNA Primer primase 2 Polymerase III Lagging strand Okazaki Fragments 1 RNA primer replaced by polymerase I & gap is sealed by ligase Ramya Rani Vootla

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DNA ligase seals the gaps between Okazaki fragments with a phosphodiester bond (Fig. 3.7) Ramya Rani Vootla

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Peter J. Russell, iGenetics : Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Fig. 3.6a, b Model for the events occurring around a single replication fork of the E. coli chromosome Ramya Rani Vootla

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Peter J. Russell, iGenetics : Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Fig. 3.6c-e Model for the events occurring around a single replication fork of the E. coli chromosome Ramya Rani Vootla

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Concepts and terms to understand: Why are gyrase and helicase required? The difference between a template and a primer ? The difference between primase and polymerase ? What is a replication fork and how many are there? Why are single-stranded binding (SSB) proteins required? How does synthesis differ on leading strand and lagging strand ? Which is continuous and semi-discontinuous ? What are Okazaki fragments ? Ramya Rani Vootla

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Replication of circular DNA in E. coli (3.10): Two replication forks result in a theta-like ( ) structure. As strands separate, positive supercoils form elsewhere in the molecule. Topoisomerases relieve tensions in the supercoils, allowing the DNA to continue to separate. Ramya Rani Vootla

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Rolling circle model of DNA replication (3.12): Common in several bacteriophages including . Begins with a nick at the origin of replication. 5’ end of the molecule is displaced and acts as primer for DNA synthesis. Can result in a DNA molecule many multiples of the genome length (and make multiple copies quickly). During viral assembly the DNA is cut into individual viral chromosomes. Ramya Rani Vootla

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DNA replication in eukaryotes: Copying each eukaryotic chromosome during the S phase of the cell cycle presents some challenges: Major checkpoints in the system Cells must be large enough, and the environment favorable. Cell will not enter the mitotic phase unless all the DNA has replicated. Chromosomes also must be attached to the mitotic spindle for mitosis to complete. Checkpoints in the system include proteins call cyclins and enzymes called cyclin-dependent kinases (Cdks). Ramya Rani Vootla

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Eukaryotic enzymes: Five DNA polymerases from mammals. Polymerase  ( alpha): nuclear, DNA replication, no proofreading Polymerase  ( beta): nuclear, DNA repair, no proofreading Polymerase  ( gamma): mitochondria, DNA repl., proofreading Polymerase  ( delta): nuclear, DNA replication, proofreading Polymerase  ( epsilon): nuclear, DNA repair (?), proofreading Different polymerases for nucleus and mtDNA Some proofread; others do not. Some used for replication; others for repair. Ramya Rani Vootla

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Each eukaryotic chromosome is one linear DNA double helix Average ~10 8 base pairs long With a replication rate of 2 kb/minute, replicating one human chromosome would require ~35 days. Solution ---> DNA replication initiates at many different sites simultaneously. Rates are cell specific! Fig. 3.17 Ramya Rani Vootla

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What about the ends (or telomeres) of linear chromosomes? DNA polymerase/ligase cannot fill gap at end of chromosome after RNA primer is removed. Big problem---If this gap is not filled, chromosomes would become shorter each round of replication! Solution: Eukaryotes have tandemly repeated sequences at the ends of their chromosomes. Telomerase (composed of protein and RNA complementary to the telomere repeat) binds to the terminal telomere repeat and catalyzes the addition of of new repeats. Compensates by lengthening the chromosome. Absence or mutation of telomerase activity results in chromosome shortening and limited cell division. Ramya Rani Vootla

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Peter J. Russell, iGenetics : Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Fig. 3.19 Synthesis of telomeric DNA by telomerase Ramya Rani Vootla