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CHAPTER 13

CHAPTER 13. DNA Replication and Repair. Introduction. Reproduction is a property of all organisms. DNA duplicates by a process called DNA replication . The DNA replication machinery is also used for DNA repair. 13.1 DNA Replication (1).

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CHAPTER 13

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  1. CHAPTER 13 DNA Replication and Repair

  2. Introduction • Reproduction is a property of all organisms. • DNA duplicates by a process called DNA replication. • The DNA replication machinery is also used for DNA repair.

  3. 13.1 DNA Replication (1) • DNA replication takes place by separation of the strands of the double helix, and synthesis of two daughter strands complementary to the two parental templates.

  4. DNA Replication (2) • Semiconservative Replication • DNA replication is called semiconservative because half of the parent structure is retained in each of the daughter duplexes. • This model of DNA replication took over the other tow models previously considered: conservative and dispersive.

  5. Three alternate schemes of replication

  6. Three alternate schemes of replication

  7. Three alternate schemes of replication

  8. DNA Replication (3) • The Messelson and Stahl experiments supported the semiconservative model of replication in bacterial cells. • Semiconservative replication was later demonstrated in eukaryotic cells.

  9. Experimental demonstration of semiconservative DNA replication in bacteria

  10. Experimental demonstration of semiconservative DNA replication in eukaryotes

  11. DNA Replication (4) • Replication in Bacterial Cells • Temperature-sensitive (ts) mutants were used to identify the genes of replication. • Replication can be studied using in vitro systems reconstituted from purified cellular compounds.

  12. DNA Replication (5) • Replication Forks and Bidirectional Replication • Replication starts at the origin site, where a number of proteins bind to initiate replication. • Replication proceeds bidirectionally. • Replication forks are points where a pair of replicating segments come together and join the nonreplicated segments.

  13. Model of a bacterial chromosome undergoing bidirectional replication

  14. DNA Replication (6) • Unwinding the Duplex and Separating the Strands • Tension is built up as DNA begins the unwinding process, and the DNA becmes positively supercoiled. • DNA gyrase (topoisomerase II) relieves the tension by changing the DNA into negatively supercoiled DNA.

  15. The unwinding problem

  16. DNA Replication (7) • The Properties of DNA Polymerases • DNA polymerase is responsible for synthesizing new DNA strands from a DNA template. • DNA polymerase requires a primer which provides the 3’ hydroxyl terminus on which to add new nucleotides. • Polymerization occurs in the 5’-to-3’ direction. • None of the three DNA polymerases in bacteria can initiate DNA chains.

  17. Templates and nontemplates for DNA polymerase activity

  18. DNA Replication (8) • Semidiscontinuous Replication • Both daughter strands are synthesized simultaneously. • The leading strand (in the direction of the replication fork movement) is synthesized continuously. • The lagging strand (in the opposite direction of the replication fork movement) is synthesized discontinuously.

  19. The incorporation of nucleotides onto the 3’ end of a growing strand by DNA polymerase

  20. The two strands of a double helix are synthesized by a different sequence of events

  21. DNA Replication (9) • The lagging strand is constructed of small Okazaki fragments, which are joined by DNA ligase.

  22. DNA Replication (10) • Primase is an RNA polymerase that assembles short RNA primers. • These primers are later removed and the gaps are sealed.

  23. DNA Replication (11) • The Machinery Operating at the Replication Fork • Helicase and single-stranded DNA-binding (SSB) proteins unwind the parental duplex and separate the two strands. • Primase and helicase form a “primosome”, which processively moves along the lagging-strand template. • A single replisome synthesizes both leading and lagging strands.

  24. The role of DNA helicase, SSB proteins, and primase at the replication fork

  25. The role of DNA helicase, SSB proteins, and primase at the replication fork

  26. DNA Replication (12) • The Structure and Functions of DNA Polymerases • DNA polymerase III is the primary replication enzyme. • DNA polymerase III holoenzyme contains various subunits having different functions in the replication process. • By forming a β clamp, one of the components maintains an association between the polymerase and the DNA template.

  27. Replication in E. coli by DNA polymerases working together as part of a complex

  28. DNA Replication (13) • As long as it is attached to a β “sliding clamp”, DNA polymerase can move processively from one nucleotide to the next. • The assembly of the β clamp around the DNA requires a clamp loader, which is part of the DNA polymerase III holoenzyme.

  29. Schematic representation ofDNA polymerase III

  30. The β sliding clamp and clamp loader

  31. DNA Replication (14) • Exonuclease Activities of DNA Polymerases • DNA polymerase I is involved in DNA repair and also removes RNA primers and replaces them with DNA. • Exonucleases degrade nucleic acids by removing 5’ or 3’ terminal nucleotides.

  32. The exonuclease activities ofDNA polymerase I

  33. DNA Replication (15) • Ensuring High Fidelity during DNA Replication • The error rate of incorporation of an incorrect nucleotide during DNA replication is the spontaneous mutation rate. • Incorporation of a particular nucleotide onto the end of growing strand depends upon the geometry of the base pair.

  34. Geometry of proper andmismatched base pairs

  35. DNA Replication (16) • Ensuring high fidelity (continued) • During proofreading, mismatched bases are excised. • Careful selection of the nucleotide, proofreading, and mismatch repair account for low error rates in replication (about 10–9). • Replication is rapid (~103 nucleotides/sec).

  36. Activation of the 3’  5’ exonuclease activity of DNA polymerase I

  37. DNA Replication (17) • Replication in Eukaryotic Cells • Replication is eukaryotes is not as well understood as replication in bacteria. Some advances include: • Using mutant yeast cells unable to produce specific gene products for replication. • Development of in vitro systems where replication can occur in cellular extracts or mixtures of purified proteins.

  38. DNA Replication (18) • Initiation of Replication in Eukaryotic Cells • Eukaryotes replicate their genome in small portions (replicons). • Initiation of DNA synthesis in a replicon is regulated.

  39. DNA Replication (19) • Origins of replication identified in yeast cells are called autonomous replicating sequences (ARS). • A multiprotein origin recognition complex (ORC) is assembled at the ARS. • Replication in mammalian cells has been more difficult to study.

  40. Replication of a yeast replicon

  41. DNA Replication (20) • Restricting Replication to Once Per Cell Cycle • Replication origins pass through different states so that they only replicate their DNA once during a cell cycle. • Origin of replication bound by an ORC. • Proteins called “licensing factors” bind to the origin. • Activation factors bind to the chromosomes and induce “licensed” origins to begin replication.

  42. DNA Replication (21) • The Eukaryotic Replication Fork • Replication activities are similar in eukaryotes and prokaryotes. • There are several DNA polymerases in eukaryotes. • Eukaryotic DNA polymerases elongate in the 5’-to-3’ direction and require a primer; some have 3’-to-5’ exonuclease activity.

  43. Some Proteins Required for EukaryoticDNA Replication

  44. Schematic view of the major components of the eukaryotic replication fork

  45. DNA Replication (22) • Replication and Nuclear Structure • The replication machinery is stationary in the nuclear matrix. • Replication forks are located within sites called replication foci. • The clustering of replication forks may provide a mechanism for coordinating replication of adjacent replicons on individual chromosomes.

  46. The involvement of the nuclear matrixin DNA replication

  47. Demonstration that replication activities are confined to distinct sites

  48. DNA Replication (23) • Chromatin Structure and Replication • The assembly of DNA into nucleosomes is a rapid event. • Histones remain intact during replication and old and new histones are distributed randomly between the two daughter duplexes. • The assembly of nucleosomes is facilitated by a network of accessory proteins.

  49. The distribution of histone core complexes to daughter cells following replication

  50. 13.2 DNA Repair (1) • DNA repair is essential for cell survival. • DNA is the cell molecule most susceptible to environmental damage. • Ionizing radiation, common chemicals, UV radiation and thermal energy create spontaneous alteration (lesions) in DNA. • Cells have a number of mechanisms to repair genetic damage.

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