biochemistry 2/e - garrett & grisham copyright © 1999 by harcourt brace & company chapter...

Biochemistry 2/e - Garrett & Grisham

Copyright © 1999 by Harcourt Brace & Company

Chapter 30

DNA Replication and Repairto 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



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



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.



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



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



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



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



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



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



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!



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"



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



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



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



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



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



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"



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



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



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



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



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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