Distinct mechanism of cis-syn thymine dimer bypass by Dpo4 and DNA polymerase η

Robert E. Johnson, Louise Prakash, Satya Prakash

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

UV-light-induced cyclobutane pyrimidine dimers (CPDs) present a severe block to synthesis by replicative DNA polymerases (Pols), whereas Polη promotes proficient and error-free replication through CPDs. Although the archael Dpo4, which, like Polη, belongs to the Y family of DNA Pols, can also replicate through a CPD, it is much less efficient than Polη. The x-ray crystal structure of Dpo4 complexed with either the 3′-thymine (T) or the 5′ T of a cis-syn TT dimer has indicated that, whereas the 3′ T of the dimer forms a Watson-Crick base pair with the incoming dideoxy ATP, the 5′ T forms a Hoogsteen base pair with the dideoxy ATP in syn conformation. Based upon these observations, a similar mechanism involving Hoogsteen base pairing of the 5′ T of the dimer with the incoming A has been proposed for Polη. Here we examine the mechanisms of CPD bypass by Dpo4 and Polη using nucleotide analogs that specifically disrupt the Hoogsteen or Watson-Crick base pairing. Our results show that both Dpo4 and Polη incorporate dATP opposite the 5′ T of the CPD via Watson-Crick base pairing and not by Hoogsteen base pairing. Furthermore, opposite the 3′ T of the dimer, the two Pols differ strikingly in the mechanisms of dATP incorporation, with Dpo4 incorporating opposite an abasic-like intermediate and Polη using the normal Watson-Crick base pairing. These observations have important implications for the mechanisms used for the inefficient vs. efficient bypass of CPDs by DNA Pols.

Original languageEnglish (US)
Pages (from-to)12359-12364
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume102
Issue number35
DOIs
StatePublished - Aug 30 2005

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Pyrimidine Dimers
DNA-Directed DNA Polymerase
Base Pairing
Adenosine Triphosphate
Thymine
Ultraviolet Rays
Nucleotides
X-Rays

Keywords

  • Cyclobutane pyrimidine dimer
  • Hoogsteen base pairing
  • Translesion DNA synthesis
  • Watson-Crick base pairing

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

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title = "Distinct mechanism of cis-syn thymine dimer bypass by Dpo4 and DNA polymerase η",
abstract = "UV-light-induced cyclobutane pyrimidine dimers (CPDs) present a severe block to synthesis by replicative DNA polymerases (Pols), whereas Polη promotes proficient and error-free replication through CPDs. Although the archael Dpo4, which, like Polη, belongs to the Y family of DNA Pols, can also replicate through a CPD, it is much less efficient than Polη. The x-ray crystal structure of Dpo4 complexed with either the 3′-thymine (T) or the 5′ T of a cis-syn TT dimer has indicated that, whereas the 3′ T of the dimer forms a Watson-Crick base pair with the incoming dideoxy ATP, the 5′ T forms a Hoogsteen base pair with the dideoxy ATP in syn conformation. Based upon these observations, a similar mechanism involving Hoogsteen base pairing of the 5′ T of the dimer with the incoming A has been proposed for Polη. Here we examine the mechanisms of CPD bypass by Dpo4 and Polη using nucleotide analogs that specifically disrupt the Hoogsteen or Watson-Crick base pairing. Our results show that both Dpo4 and Polη incorporate dATP opposite the 5′ T of the CPD via Watson-Crick base pairing and not by Hoogsteen base pairing. Furthermore, opposite the 3′ T of the dimer, the two Pols differ strikingly in the mechanisms of dATP incorporation, with Dpo4 incorporating opposite an abasic-like intermediate and Polη using the normal Watson-Crick base pairing. These observations have important implications for the mechanisms used for the inefficient vs. efficient bypass of CPDs by DNA Pols.",
keywords = "Cyclobutane pyrimidine dimer, Hoogsteen base pairing, Translesion DNA synthesis, Watson-Crick base pairing",
author = "Johnson, {Robert E.} and Louise Prakash and Satya Prakash",
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T1 - Distinct mechanism of cis-syn thymine dimer bypass by Dpo4 and DNA polymerase η

AU - Johnson, Robert E.

AU - Prakash, Louise

AU - Prakash, Satya

PY - 2005/8/30

Y1 - 2005/8/30

N2 - UV-light-induced cyclobutane pyrimidine dimers (CPDs) present a severe block to synthesis by replicative DNA polymerases (Pols), whereas Polη promotes proficient and error-free replication through CPDs. Although the archael Dpo4, which, like Polη, belongs to the Y family of DNA Pols, can also replicate through a CPD, it is much less efficient than Polη. The x-ray crystal structure of Dpo4 complexed with either the 3′-thymine (T) or the 5′ T of a cis-syn TT dimer has indicated that, whereas the 3′ T of the dimer forms a Watson-Crick base pair with the incoming dideoxy ATP, the 5′ T forms a Hoogsteen base pair with the dideoxy ATP in syn conformation. Based upon these observations, a similar mechanism involving Hoogsteen base pairing of the 5′ T of the dimer with the incoming A has been proposed for Polη. Here we examine the mechanisms of CPD bypass by Dpo4 and Polη using nucleotide analogs that specifically disrupt the Hoogsteen or Watson-Crick base pairing. Our results show that both Dpo4 and Polη incorporate dATP opposite the 5′ T of the CPD via Watson-Crick base pairing and not by Hoogsteen base pairing. Furthermore, opposite the 3′ T of the dimer, the two Pols differ strikingly in the mechanisms of dATP incorporation, with Dpo4 incorporating opposite an abasic-like intermediate and Polη using the normal Watson-Crick base pairing. These observations have important implications for the mechanisms used for the inefficient vs. efficient bypass of CPDs by DNA Pols.

AB - UV-light-induced cyclobutane pyrimidine dimers (CPDs) present a severe block to synthesis by replicative DNA polymerases (Pols), whereas Polη promotes proficient and error-free replication through CPDs. Although the archael Dpo4, which, like Polη, belongs to the Y family of DNA Pols, can also replicate through a CPD, it is much less efficient than Polη. The x-ray crystal structure of Dpo4 complexed with either the 3′-thymine (T) or the 5′ T of a cis-syn TT dimer has indicated that, whereas the 3′ T of the dimer forms a Watson-Crick base pair with the incoming dideoxy ATP, the 5′ T forms a Hoogsteen base pair with the dideoxy ATP in syn conformation. Based upon these observations, a similar mechanism involving Hoogsteen base pairing of the 5′ T of the dimer with the incoming A has been proposed for Polη. Here we examine the mechanisms of CPD bypass by Dpo4 and Polη using nucleotide analogs that specifically disrupt the Hoogsteen or Watson-Crick base pairing. Our results show that both Dpo4 and Polη incorporate dATP opposite the 5′ T of the CPD via Watson-Crick base pairing and not by Hoogsteen base pairing. Furthermore, opposite the 3′ T of the dimer, the two Pols differ strikingly in the mechanisms of dATP incorporation, with Dpo4 incorporating opposite an abasic-like intermediate and Polη using the normal Watson-Crick base pairing. These observations have important implications for the mechanisms used for the inefficient vs. efficient bypass of CPDs by DNA Pols.

KW - Cyclobutane pyrimidine dimer

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KW - Translesion DNA synthesis

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