Chapter 13 DNA Replication

1. Chapter 13DNA Replication

2. 13.1 Introduction • 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.

3. 13.1 Introduction • 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. Figure 13.01: Semiconservative replication synthesizes two new strands of DNA.

4. 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.

5. 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. 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.

6. 13.3 DNA Polymerases Have Various Nuclease Activities • DNA polymerase I has a unique 5′ and 3′ exonuclease activity.

7. 13.4 DNA Polymerases Control the Fidelity of Replication • 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.

8. 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. 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.

9. 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. Photo courtesy of Charles Richardson and Thomas Ellenberger, Washington University School of Medicine. 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. Adapted from Protein Data Bank 1KFD. L. S. Breese, J. M. Friedman, and T. A. Steitz, Biochemistry 32 (1993): 14095-14101.

10. 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.

11. 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. Figure 13.09: A hexameric helicase moves along one strand of DNA.

12. 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. 13.8 Priming Is Required to Start DNA Synthesis • The priming end can be provided by an RNA primer, a nick in DNA, or a priming protein. Figure 13.11: There are several methods for providing the free 3′–OH end that DNA polymerases require to initiate DNA synthesis.

14. 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. • E. coli has two types of priming reactions, which occur at the bacterial origin (oriC) and the φX174 origin.

15. 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.

16. 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.

17. 13.10 DNA Polymerase Holoenzyme Consists of Subcomplexes • The E. coli replicase DNA polymerase III is a 900 kD complex with a dimeric structure. • Each monomeric unit has a catalytic core, a dimerization subunit, and a processivity component.

18. 13.10 DNA Polymerase Holoenzyme Consists of Subcomplexes • 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.

19. 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.

20. 13.11 The Clamp Controls Association of Core Enzyme with DNA • The helicase DnaB is responsible for interacting with the primaseDnaG to initiate each Okazaki fragment. Figure 13.18: Core polymerase and the β clamp dissociate at completion of Okazaki fragment synthesis and reassociate at the beginning. Figure 13.17: Each catalytic core of Pol III synthesizes a daughter strand. DnaB is responsible for forward movement at the replication fork.

21. 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.

22. 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.

23. 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. • DNA polymerase ε elongates the leading strand and a second DNA polymerase δ elongates the lagging strand.

24. 13.13 Separate Eukaryotic DNA Polymerases Undertake Initiation and Elongation Figure 13.23: Three different DNA polymerases make up the eukaryote replication fork.

25. 13.14 Lesion Bypass Requires Polymerase Replacement • A replication fork stalls when it arrives at damaged DNA. 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.

26. 13.14 Lesion Bypass Requires Polymerase Replacement • After the damage has been repaired, the primosome is required to reinitiate replication. Figure 13.26: The primosome is required to restart a stalled replication fork after the DNA has been repaired.

27. 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.