Biochemistry 2e Garrett Grisham Chapter 30 DNA Replication

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

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

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

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

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

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

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

Biochemistry 2/e - Garrett & Grisham 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 k. D) monomer In addition to 5'-3' polymerase, it also has 3'-5' exonuclease and 5'-3' exonuclease activities Copyright © 1999 by Harcourt Brace & Company

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

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

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

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

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

Biochemistry 2/e - Garrett & Grisham 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: Dna. A protein binds to repeats in ori, initiating strand separation and Dna. B, a helicase delivered by Dna. C, further unwinds. Primase then binds and constructs the RNA primer Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Replication Mechanism II Elongation and Termination • Elongation involves Dna. B 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 Copyright © 1999 by Harcourt Brace & Company

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

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

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

Biochemistry 2/e - Garrett & Grisham Reverse Transcriptase • Primer required, but a strange one - a t. RNA molecule that the virus captures from the host • RT transcribes the RNA template into a complementary DNA (c. DNA) to form a DNA: RNA hybrid • All RNA tumor viruses contain a reverse transcriptase Copyright © 1999 by Harcourt Brace & Company

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

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

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

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