Chapter 6: DNA Replication and Telomere Maintenance

1. Chapter 6: DNA Replication and Telomere Maintenance

2. It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material. James D. Watson and Francis Crick, Nature (1953), 171:737

3. 6.1 Introduction

4. DNA replication involves: • The melting apart of the two strands of the double helix followed by the polymerization of new complementary strands. • Decisions of when, where, and how to initiate replication to ensure that only one complete and accurate copy of the genome is made before a cell divides.

5. 6.2 Early insights into the mode of bacterial DNA replication

6. Three possible modes of replication hypothesized based on Watson and Crick’s model: • Semiconservative • Conservative • Dispersive

7. The Meselson-Stahl experiment • 1958 experiment designed to distinguish between semiconservative, conservative, and dispersive replication. • Results were consistent only with semiconservative replication.

8. Visualization of replicating bacterial DNA • Semiconservative mechanism of DNA replication visually verified by J. Cairns in 1963 using autoradiography. • Bidirectional replication of the E. coli chromosome. • One origin of replication. • Replication intermediates are termed theta () structures.

9. 6.3 DNA polymerases are the enzymes that catalyze DNA synthesis from 5′ to 3′

10. DNA polymerases • Can only add nucleotides in the 5′→3′ direction. • Cannot initiate DNA synthesis de novo. • Require a primer with a free 3′-OH group at the end.

11. Deoxynucleoside 5′ triphosphates (dNTPs) are added one at a time to the 3′ hydroxyl end of the DNA chain. • The dNTP added is determined by complementary base pairing. • As phosphodiester bonds form, the two terminal phosphates are lost, making the reaction essentially irreversible.

12. Problem • DNA polymerases can only add nucleotides from 5′→3′ but, the two strands of the double helix are antiparallel. Solution • Semidiscontinuous replication.

13. Semidiscontinuous DNA replication • Major form of replication in eukaryotic nuclear DNA, some viruses, and bacteria.

14. Leading strand synthesis is continuous • Once primed, continuous replication is possible on the 3′→ 5′ template strand (leading strand). • Leading strand synthesis occurs in the same direction as movement of the replication fork.

15. Leading strand synthesis is continuous • Discontinuous replication occurs on the 5′→3′ template strand (lagging strand). • DNA is copied in short segments called “Okazaki fragments” moving in the opposite direction to the replication fork. • Repetition of primer synthesis and formation of Okazaki fragments.

16. Synthesis of both strands occurs concurrently • Nucleotides are added to the leading and lagging strands at the same time and rate. • Two DNA polymerases, one for each strand.

17. Fundamental features of DNA replication are conserved from E. coli to humans. • 1984: A cell-free system allowed scientists to make progress in studying replication in eukaryotic cells. • Model system: Simian virus 40 (SV40) replication.

18. 6.4 Multi-protein machines mediate bacterial DNA replication

19. Bacterial DNA polymerases have multiple functions DNA polymerase I • Primer removal, gap filling between Okazaki fragments, and nucleotide excision repair pathway. • Two subunits: Klenow fragment has 5′→3′ polymerase activity; other subunit has both 3′→5′ and 5′→3′ exonuclease activity. • Unique ability to start replication at a nick in the DNA sugar-phosphate backbone. • Used extensively in molecular biology research.

20. DNA polymerase III • Main replicative polymerase. DNA polymerase II • Involved in DNA repair mechanisms. DNA polymerases IV and V • Mediate translesion synthesis (see Chapter 7).

21. Initiation of replication • An origin of replication is a site on chromosomal DNA where a bidirectional replication fork initiates or “fires.” • Most bacteria have a single, well-defined origin (e.g. oriC in E. coli) • Some Archaea have as many as three origins (e.g. Sulfolobus). • Usually A-T rich. • In E. coli the initiator protein DnaA can only bind to negatively supercoiled origin DNA.

22. Replication is mediated by the replisome Major parts of this multi-protein machine are: • A helicase which unwinds the parental double helix. • Two molecules of DNA polymerase III. • A primase that initiates lagging strand Okazaki fragments.

23. Major parts of this multi-protein machine, cont: • Two sliding clamps that tether DNA polymerase to the DNA. • A clamp loader that uses ATP to open and close the sliding clamps around the DNA. • Single-strand DNA binding proteins (SSB) that protect the DNA from nuclease attack.

24. Lagging strand synthesis by the replisome: • As the replication fork advances, the lagging strand polymerase remains associated with the replisome forming a loop. • The loop grows until the Okazaki fragment is complete. • DNA polymerase III is released.

25. New clamps are assembled; DNA polymerase III hops aboard to make the next Okazaki fragment. • This process occurs around the circular genome until the replication forks meet. • In E. coli, the replication forks meet at a terminus region containing sequence-specific replication arrest sites.

26. DNA polymerase I removes the RNA primers and replaces them with complementary dNTPs. • DNA ligase catalyzes the formation of a phosphodiester bond between adjacent Okazaki fragments.

27. Movement of the replication fork machinery results in: • Positive supercoiling ahead of the fork. • Negative supercoiling in the wake of the fork. • Torsional strain that could inhibit fork movement is relieved by DNA topoisomerase.

28. Topoisomerases relax supercoiled DNA Topoisomers are forms of DNA that have the same sequence but differ in: • linkage number • mobility in an electrophoresis gel Topoisomerases are enzymes that convert (isomerize) one topoisomer of DNA to another by changing the linking number (L).

29. Type I topoisomerases cause transient single-stranded breaks in DNA • Type 1A only relax negative supercoils. • Type 1B can relax both negative and positive supercoils. • Do not require ATP.

30. Type II topoisomerases cause transient double-stranded breaks in DNA • Relax both negative and positive supercoils. • Unknot or decatenate entangled DNA molecules. • Usually ATP-dependent. • Bacterial “gyrase” can introduce negative supercoils.

31. Is leading strand synthesis really continuous? • DNA polymerase III can be blocked by a damaged site on the template DNA. • Sometimes DNA polymerase collides with RNA polymerase and is stalled. • In both cases, replication can be jumpstarted on the leading strand by formation of a new primer at the replication fork.

32. 6.5 Multi-protein machines trade places during eukaryotic DNA replication

33. Eukaryotic origins of replication • Internal sites on linear chromosomes. • Mice have 25,000 origins, spanning ~150 kb each. • Humans have 10,000 to 100,000 origins.

34. In the budding yeast Saccharomyces cerevisiae there is a consensus sequence called an autonomous replicating sequence (ARS). • Mammalian origin sequences are usually AT rich but lack a consensus sequence.

35. Mapping eukaryotic DNA replication origins • Analysis by two-dimensional agarose gel electrophoresis. • Other techniques allow detection of the start site for DNA synthesis at the nucleotide level. • Data suggest that there is a single defined start point.

36. Selective activation of origins of replication • The overall rate of replication is largely determined by the number of origins used and the rate at which they initiate. • During early embryogenesis, origins are uniformly activated. • At the mid-blastula transition, replication becomes restricted to specific origin sites.

37. Replication factories • Replication forks are clustered in “replication factories.” • Forty to many hundreds of forks are active in each factory. • Shown by a pulse-chase technique using BrdU labeling of cells in S-phase and detection with anti-BrdU antibodies.

38. Histone removal at origins of replication • Histone modification and chromatin remodeling factors. • Disassembly of the nucleosomes. • Template DNA is accessible to the replication machinery.

39. Prereplication complex formation and replication licensing • DNA replication is restricted to S phase of the cell cycle. • Origin selection is a separate step from initiation. • Formation of a prereplication complex. • Prevents overreplication of the genome.

40. Assembly of the origin recognition complex • The ATP-dependent origin recognition complex (ORC) binds origin sequences. • Recruits Cdc6 and Mcm proteins. • The SV40 T antigen functions as a viral ORC.

41. The naming of genes involved in DNA replication • Many genes first characterized in the yeast Saccharomyces cerevisiae. • Mutations that affect the cell cycle were isolated as conditional, temperature-sensitive mutants. • At the permissive temperature, the gene product can function. • At the restrictive temperature, mutant yeast accumulate at a particular point in the cell cycle.

42. Assembly of the replication licensing complex • In association with Cdc6 and Cdt1, ORC loads the licensing protein complex, Mcm2-7. • Mcm2-7 is a hexameric complex with helicase activity. • Only licensed origins containing Mcm2-7 can initiate a pair of replication forks.

43. ATP hydrolysis by ORC stimulates prereplication complex assembly. • Prereplication complex assembly is inhibited when ORC is bound by a nonhydrolyzable analog of ATP (ATP-S)

44. Regulation of the replication licensing system by CDKs • Replication licensing is regulated by the activity levels of cyclin-dependent kinases (CDKs). • For catalysis, CDKs must associate with a cyclin. • Cyclins accumulate gradually during interphase and are abruptly destroyed during mitosis.

45. ORC, Cdc6, Cdt1, and Mcm2-7 are downregulated by high CDK activity. • The mode of downregulation differs for each protein. • No further Mcm2-7 can be loaded onto origins in S phase, G2, and early mitosis when CDK activity is high.

46. Duplex unwinding at replication forks • DNA helicases are enzymes that use the energy of ATP to melt the DNA duplex. • They catalyze the transition from double-stranded to single-stranded DNA in the direction of the moving replication fork. • Mcm2-7 helicase is bound to the leading strand template and moves 3′→5′.

47. RNA priming of leading and lagging strand DNA synthesis • In eukaryotes, the RNA primer is synthesized by DNA polymerase (pol)  and its associated primase activity. • The pol /primase enzyme synthesizes a short strand of 10 bases of RNA, followed by 20-30 bases of initiator DNA (iDNA).

48. Polymerase switching • A key feature of the replication process is the ordered hand-off, or “trading places”, from one protein complex to another. • Polymerase switching: The hand-off of the DNA template from one polymerase to another.

49. Elongation of leading strands and lagging strands At least 14 different eukaryotic DNA polymerases • Chromosomal DNA replication DNA pol , pol , pol  • Mitochondrial DNA replication DNA pol  • Repair processes All the rest (Chapter 7)

50. Leading strand: switch from DNA polymerase  to pol  • Lagging strand: switch from pol  to pol  • Polymerase switching is regulated by PCNA.