Chapter 13 DNA Replication Jocelyn E. Krebs

Figure 13.CO: A type II topoisomerase acting on a knotted or supercoiled DNA substrate. Cleaved DNA is shown in green. The DNA that is being passed through the break is viewed end-on. Photo courtesy of James M. Berger, California Institute for Quantitative Biology, University of California, Berkeley.

13.1 Introduction DNA replication: initiation, elongation, and termination Initiation: recognize origin by large protein complex, create replication bubble, replication fork formation Elongation: replisome moves along DNA strand termination topoisomerase – An enzyme that changes the number of times the two strands in a closed DNA molecule cross each other. It does this by cutting the DNA, passing DNA through the break, and resealing the DNA. replisome – The multiprotein structure that assembles at the bacterial replication fork to undertake synthesis of DNA. It contains DNA polymerase and other enzymes.

conditional lethal – A mutation that is lethal under one set of conditions, but not lethal under a second set of permissive conditions, such as temperature (eg. temp sensitive mutant). Figure 13.01: Semiconservative replication synthesizes two new strands of DNA.

13.2 DNA Polymerases Are the Enzymes That Make DNA DNA is synthesized in both semiconservative replication and DNA repair reactions. Figure 13.01: Semiconservative replication synthesizes two new strands of DNA. Figure 13.02: Repair synthesis replaces a short stretch of one strand of DNA containing a damaged base.

13.2 DNA Polymerases Are the Enzymes That Make DNA A bacterium or eukaryotic cell has several different DNA polymerase enzymes. One bacterial DNA polymerase undertakes semiconservative replication; the others are involved in repair reactions. Holoenzyme Figure 13.03: DNA is synthesized by adding nucleotides to the 3′–OH end of the growing chain, so that the new chain grows in the 5′→3′ direction.

Figure 13. 04: Only one DNA polymerase is the replication enzyme Figure 13.04: Only one DNA polymerase is the replication enzyme. The others participate in repair of damaged DNA, restarting stalled replication forks, or bypassing damage in DNA.

13.3 DNA Polymerases Have Various Nuclease Activities DNA polymerase I has a unique 5′ and 3′ exonuclease activity.  proofreading function (error control system) 103kD two parts ; klenow fragment (68kD), in vitro synthesis Small fragment (35kD); 5-3 exonuclease (remove -10 bp) Lagging strand

13.4 DNA Polymerases Control the Fidelity of Replication General error frequency: 108-1010 1 error/genome/1000 bacteria proofreading – A mechanism for correcting errors in DNA synthesis that involves scrutiny of individual units after they have been added to the chain. processivity – The ability of an enzyme to perform multiple catalytic cycles with a single template instead of dissociating after each cycle.  replication slippage

13.4 DNA Polymerases Control the Fidelity of Replication DNA polymerases often have a 3′–5′ exonuclease activity that is used to excise incorrectly paired bases. The fidelity of replication is improved by proofreading by a factor of ~100. Error-prone DNA pol (DNA pol IV) Pol III; exonuclease activity in e subunit Figure 13.05: Bacterial DNA polymerases scrutinize the base pair at the end of the growing chain and excise the nucleotide added in the case of a misfit.

13.5 DNA Polymerases Have a Common Structure Many DNA polymerases have a large cleft composed of three domains that resemble a hand. DNA lies across the palm in a groove created by the fingers and thumb. Figure 13.06: The structure of the Klenow fragment from E. coli DNA polymerase I. Figure 13.07: The crystal structure of phage T7 DNA polymerase shows that the template strand takes a sharp turn that exposes it to the incoming nucleotide. Photo courtesy of Charles Richardson and Thomas Ellenberger, Washington University School of Medicine. Adapted from Protein Data Bank 1KFD. L. S. Breese, J. M. Friedman, and T. A. Steitz, Biochemistry 32 (1993): 14095-14101.

Thumb (processivity) Finger (Template position) Palm (red) Catalytic activity Figure 13.06: The structure of the Klenow fragment from E. coli DNA polymerase I. Adapted from Protein Data Bank 1KFD. L. S. Breese, J. M. Friedman, and T. A. Steitz, Biochemistry 32 (1993): 14095-14101.

Figure 13.07: The crystal structure of phage T7 DNA polymerase shows that the template strand takes a sharp turn that exposes it to the incoming nucleotide. Photo courtesy of Charles Richardson and Thomas Ellenberger, Washington University School of Medicine.

13.6 The Two New DNA Strands Have Different Modes of Synthesis The DNA polymerase advances continuously when it synthesizes the leading strand (5′–3′), but synthesizes the lagging strand by making short fragments (Okazaki fragments) that are subsequently joined together. semidiscontinuous replication – The mode of replication in which one new strand is synthesized continuously while the other is synthesized discontinuously. Figure 13.08: The leading strand is synthesized continuously, whereas the lagging strand is synthesized discontinuously.

13.7 Replication Requires a Helicase and Single-Strand Binding Protein Replication requires a helicase to separate the strands of DNA using energy provided by hydrolysis of ATP. A single-stranded binding protein (SSB) is required to maintain the separated strands. Helicase cannot initiate DNA melting, it just extend and expand DNA separation In bacteria, 12 different helicase, which works as hexamer Figure 13.09: A hexameric helicase moves along one strand of DNA.

13.8 Priming Is Required to Start DNA Synthesis All DNA polymerases require a 3′–OH priming end to initiate DNA synthesis. Figure 13.10: A DNA polymerase requires a 3′–OH end to initiate replication.

13.8 Priming Is Required to Start DNA Synthesis The priming end can be provided by an RNA primer, a nick in DNA (repair), or a priming protein (virus). Primer is synthezed by primase (DNA G primase) Or pre-existed RNA (tRNA) retrovirus Figure 13.11: There are several methods for providing the free 3′–OH end that DNA polymerases require to initiate DNA synthesis.

13.8 Priming Is Required to Start DNA Synthesis For DNA replication, a special RNA polymerase called a primase synthesizes an RNA chain that provides the priming end. DnaG; 60KD, single polypeptide. 10 bp primer synthesis E. coli has two types of priming reactions, which occur at the bacterial origin (oriC; DnaG) and the φX174 origin (restart by replication fork collapse, primosome).

13.8 Priming Is Required to Start DNA Synthesis Priming of replication on double-stranded DNA always requires a replicase, SSB, and primase. DnaB is the helicase that unwinds DNA for replication in E. coli. Figure 13.12: Initiation requires several enzymatic activities, including helicases, single-strand binding proteins, and synthesis of the primer.

13.9 Coordinating Synthesis of the Lagging and Leading Strands Different enzyme units are required to synthesize the leading and lagging strands. Figure 13.13: A replication complex contains separate catalytic units for synthesizing the leading and lagging strands.

13.10 DNA Polymerase Holoenzyme Consists of Subcomplexes The E. coli replicase DNA polymerase III is a 900 kD complex with a dimeric structure. a subunit DNA polymerase e subunit 3-5 proofreading exonuclease q subunit stimulates the exonuclease t linker between two catalytic cores Each monomeric unit has a catalytic core, a dimerization subunit, and a processivity component.

One catalytic core is associated with each template strand. Clamp : binding DNA, homodimeric b-subunit ring g-complex; clamp loader, 5 protein complex A clamp loader places the processivity subunits on DNA, where they form a circular clamp around the nucleic acid. One catalytic core is associated with each template strand. Figure 13.14: DNA polymerase III holoenzyme assembles in stages, generating an enzyme complex that synthesizes the DNA of both new strands.

Clamp loading on DNA ; ATP hydrolysis by clamp loader Conformation change (b-subunit) Recruit clamp loader complex Clamp turned core enzyme binding conformation t-dimer binding to core-enzyme dimer formation

13.11 The Clamp Controls Association of Core Enzyme with DNA Ice skate: water-mediated interaction and moving

13.11 The Clamp Controls Association of Core Enzyme with DNA The core on the leading strand is processive because its clamp keeps it on the DNA. The clamp associated with the core on the lagging strand dissociates at the end of each Okazaki fragment and reassembles for the next fragment. Figure 13.16: The helicase creating the replication fork is connected to two DNA polymerase catalytic subunits, each of which is held on to DNA by a sliding clamp.

13.11 The Clamp Controls Association of Core Enzyme with DNA The helicase DnaB is responsible for interacting with the primase DnaG to initiate each Okazaki fragment. Figure 13.17: Each catalytic core of Pol III synthesizes a daughter strand. DnaB is responsible for forward movement at the replication fork. Figure 13.18: Core polymerase and the β clamp dissociate at completion of Okazaki fragment synthesis and reassociate at the beginning.

Figure 13.16: The helicase creating the replication fork is connected to two DNA polymerase catalytic subunits, each of which is held on to DNA by a sliding clamp.

DnaB and t-subunit binding full complex Increase DNA synthesis speed (10 fold ) Blocking the dissociation leading strand DNA pol form DNA Trombone model Figure 13.17: Each catalytic core of Pol III synthesizes a daughter strand. DnaB is responsible for forward movement at the replication fork.

leading strand pol progress lagging strand loop is larger Lagging strand synthesis is completed dissociation of b-ring by clamp loader Core enzyme is dissociated from lagging strand DnaG determine second syntheitc site (DnaB works as critical role) Figure 13.18: Core polymerase and the β clamp dissociate at completion of Okazaki fragment synthesis and reassociate at the beginning.

13.12 Okazaki Fragments Are Linked by Ligase Each Okazaki fragment starts with a primer and stops before the next fragment. In E. coli, DNA polymerase I removes the primer and replaces it with DNA. Figure 13.19: Synthesis of Okazaki fragments require priming, extension, removal of RNA primer, gap filling, and nick ligation.

13.12 Okazaki Fragments Are Linked by Ligase DNA ligase makes the bond that connects the 3′ end of one Okazaki fragment to the 5′ beginning of the next fragment. Figure 13.20: DNA ligase seals nicks between adjacent nucleotides by employing an enzyme-AMP intermediate.

Figure 13.21: Eukaryotic cells have many DNA polymerases. 13.13 Separate Eukaryotic DNA Polymerases Undertake Initiation and Elongation A replication fork has one complex of DNA polymerase α/primase and two complexes of DNA polymerase  and/or ε. The DNA polymerase α /primase complex initiates the synthesis of both DNA strands. 180 kD, B subunit-assembly, small two subunits primase (RNA pol) DNA polymerase ε elongates the leading strand and a second DNA polymerase δ elongates the lagging strand. Figure 13.21: Eukaryotic cells have many DNA polymerases.

Pol a/primase binding In Leading strand, pol e Lagging strand, pol d Polymerase switch Pol e interacted with RF-C and PCNA

Figure 13.22: Similar functions are required at all replication forks.

13.14 Lesion Bypass Requires Polymerase Replacement A replication fork stalls when it arrives at damaged DNA. Two options to avoid death Lesion bypass by error prone DNA pol recombination Figure 13.24: The replication fork stalls and may collapse when it reaches a damaged base or a nick in DNA. Arrowheads indicate 3′ ends.

Error prone DNA pol DNA pol IV and V in E.coli  Which is replaced after bypass with Pol III Eukaryotic ; 5 error prone DNA pol Replication fork halting is common event (10-50%) Figure 13.24: The replication fork stalls and may collapse when it reaches a damaged base or a nick in DNA. Arrowheads indicate 3′ ends.

Recombination Damaged DNA excision  rebuilding redundant strand  Gap filling Figure 13.25: Replication halts at damaged DNA, the damaged sequence is excised and the complementary of the other daughter duplex crosses over to repair the gap.

13.14 Lesion Bypass Requires Polymerase Replacement After the damage has been repaired, the primosome is required to reinitiate replication. (reloading DnaB) Figure 13.26: The primosome is required to restart a stalled replication fork after the DNA has been repaired.

13.15 Termination of Replication The two E. coli replication forks usually meet halfway around the circle, but there are ter sites that halt the replication fork if it advances too far. Figure 13.27: Replication termini in E. coli are located in a region between two sets of ter sites.