Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Chapter 30 DNA Replication and Repair to accompany Biochemistry, 2/e.

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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.

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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 The beta subunit dimer forms a ring around DNA Enormous processivity - 5 million bases!

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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"

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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"

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Chapter 30 DNA Replication and Repair to accompany Biochemistry, 2/e.

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Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Chapter 30 DNA Replication and Repair to accompany Biochemistry, 2/e.

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Presentation on theme: "Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Chapter 30 DNA Replication and Repair to accompany Biochemistry, 2/e."— Presentation transcript:

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