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Chapter 30

Chapter 30. DNA Replication and Repair to accompany Biochemistry, 2/e by Reginald Garrett and Charles Grisham.

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Chapter 30

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  1. Chapter 30 DNA Replication and Repair to accompany Biochemistry, 2/e by Reginald Garrett and Charles Grisham All rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida 32887-6777

  2. Outline • 30.1 DNA Replication is Semiconservative • 30.2 General Features of DNA Replication • 30.3 DNA Polymerases • 30.4 The Mechanism of DNA Replication • 30.5 Eukaryotic DNA Replication • 30.6 Telomeres and Telemerases • 30.7 Reverse Transcriptase • 30.8 DNA Repair

  3. The Dawn of Molecular Biology April 25, 1953 • Watson and Crick: "It has not escaped our notice that the specific (base) pairing we have postulated immediately suggests a possible copying mechanism for the genetic material." • The mechanism: Strand separation, followed by copying of each strand. • Each separated strand acts as a template for the synthesis of a new complementary strand.

  4. DNA Replication The Semiconservative Model • Matthew Meselson and Franklin Stahl showed that DNA replication results in new DNA duplex molecules in which one strand is from the parent duplex and the other is completely new • Study Figure 30.4 and understand the density profiles from ultracentrifugation experiments • Imagine and predict the density profiles that the conservative and dispersive models would show

  5. Features of DNA Replication • DNA replication is bidirectional • Bidirectional replication involves two replication forks, which move in opposite directions • DNA replication is semidiscontinuous • The leading strand copies continuously • The lagging strand copies in segments (Okazaki fragments) which must be joined

  6. The Enzymology of DNA Replication • If Watson and Crick were right, then there should be an enzyme that makes DNA copies from a DNA template • In 1957, Arthur Kornberg and colleagues demonstrated the existence of a DNA polymerase - DNA polymerase I • Pol I needs all four deoxynucleotides, a template and a primer - a ss-DNA (with a free 3'-OH) that pairs with the template to form a short double-stranded region

  7. DNA Polymerase I Replication occurs 5' to 3' • Nucleotides are added at the 3'-end of the strand • Pol I catalyzes about 20 cycles of polymerization before the new strand dissociates from template • 20 cycles constitutes moderate "processivity" • Pol I from E. coli is 928 aa (109 kD) monomer • In addition to 5'-3' polymerase, it also has 3'-5' exonuclease and 5'-3' exonuclease activities

  8. More on Pol I Why the exonuclease activity? • The 3'-5' exonuclease activity serves a proofreading function! It removes incorrectly matched bases, so that the polymerase can try again • See Figures 30.9 and 30.10! Notice how the newly-formed strand oscillates between the polymerase and 3'-exonuclease sites,adding a base and then checking it

  9. Even More on Pol I Nicks and Klenows.... • 5'-exonuclease activity, working together with the polymerase, accomplishes "nick translation" • Hans Klenow used either subtilisin or trypsin to cleave between residues 323 and 324, separating 5'-exonuclease (on 1-323) and the other two activities (on 324-928, the so-called "Klenow fragment”) • Tom Steitz has determined the structure of the Klenow fragment - see Figure 30.9

  10. DNA Polymerase III The "real" polymerase in E. coli • At least 10 different subunits • "Core" enzyme has three subunits - , , and  • Alpha subunit is polymerase • Epsilon subunit is 3'-exonuclease • Theta function is unknown • Thebetasubunit dimer forms a ring around DNA • Enormous processivity - 5 million bases!

  11. Features of Replication Mostly in E. coli, but many features are general • Replication is bidirectional • The double helix must be unwound - by helicases • Supercoiling must be compensated - by DNA gyrase • Replication is semidiscontinuous • Leading strand is formed continuously • Lagging strand is formed from Okazaki fragments - discovered by Tuneko and Reiji "O"

  12. More Features of Replication • Read page 994 on chemistry of DNA synthesis • DNA Pol III uses an RNA primer • A special primase forms the required primer • DNA Pol I excises the primer • DNA ligase seals the "nicks" between Okazaki fragments (See Figure 30.14 for mechanism) • See Figure 30.15 for a view of replication fork

  13. Mechanism of Replication in E. coli • The replisome consists of: DNA-unwinding proteins, the priming complex (primosome) and two equivalents of DNA • polymerase III holoenzyme • Initiation: DnaA protein binds to repeats in ori, initiating strand separation and DnaB, a helicase delivered by DnaC, further unwinds. Primase then binds and constructs the RNA primer

  14. Replication Mechanism II Elongation and Termination • Elongation involves DnaB helicase unwinding, SSB binding to keep strands separated, and DNA polymerase grinding away on both strands • Termination: the "ter" locus, rich in Gs and Ts, signals the end of replication. A Ter protein is also involved. Ter protein is a contrahelicase and prevents unwinding • Topoisomerase II (DNA gyrase) relieves supercoiling that remains

  15. Eukaryotic DNA Replication Like E. coli, but more complex • Human cell: 6 billion base pairs of DNA to copy • Multiple origins of replication: 1 per 3- 300 kbp • Several known animal DNA polymerases - see Table 30.4 • DNA polymerase alpha - four subunits, polymerase (processivity = 200) but no 3'-exonuclease • DNA polymerase beta - similar to alpha

  16. More Eukaryotic polymerases • DNA polymerase gamma - DNA-replicating enzyme of mitochondria • DNA polymerase delta has a 3'-exonuclease as well as proliferating cell nuclear antigen (PCNA) • PCNA give delta unlimited processivity and is homologous with prokaryotic pol III • DNA polymerase epsilon - highly processive, but does not have a subunit like PCNA

  17. Another Way to Make DNA RNA-Directed DNA Polymerase • 1964: Howard Temin notices that DNA synthesis inhibitors prevent infection of cells in culture by RNA tumor viruses. Temin predicts that DNA is an intermediate in RNA tumor virus replication • 1970: Temin and David Baltimore (separately) discover the RNA-directed DNA polymerase - aka "reverse trascriptase"

  18. Reverse Transcriptase • Primer required, but a strange one - a tRNA molecule that the virus captures from the host • RT transcribes the RNA template into a complementary DNA (cDNA) to form a DNA:RNA hybrid • All RNA tumor viruses contain a reverse transcriptase

  19. RT II • Three enzyme activities • RNA-directed DNA polymerase • RNase H activity - degrades RNA in the DNA:RNA hybrids • DNA-directed DNA polymerase - which makes a DNA duplex after RNase H activity destroys the viral genome • HIV therapy: AZT (or 3'-azido-2',3'- dideoxythymidine) specifically inhibits RT

  20. DNA Repair A fundamental difference from RNA, protein, lipid, etc. • All these others can be replaced, but DNA must be preserved • Cells require a means for repair of missing, altered or incorrect bases, bulges due to insertion or deletion, UV-induced • pyrimidine dimers, strand breaks or cross-links • Two principal mechanisms: mismatch repair and methods for reversing chemical damage

  21. Mismatch Repair • Mismatch repair systems scan DNA duplexes for mismatched bases, excise the mispaired region and replace it • Methyl-directed pathway of E. coli is example • Since methylation occurs post-replication, repair proteins identify methylated strand as parent, remove mismatched bases on other strand and replace them

  22. Reversing Chemical Damage • Pyrimidine dimers can be repaired by photolyase • Excision repair: DNA glycosylase removes damaged base, creating an "AP site" • AP endonuclease cleaves backbone, exonuclease removes several residues and gap is repaired by DNA polymerase and DNA ligase

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