Mechanism of nucleotide incorporation opposite a thymine-thymine dimer by yeast DNA polymerase η

M. Todd Washington, Louise Prakash, Satya Prakash

Research output: Contribution to journalArticle

63 Citations (Scopus)

Abstract

DNA polymerase η (Polη) has the unique ability to replicate through UV-light-induced cyclobutane pyrimidine dimers. Here we use pre-steady-state kinetic analyses to examine the mechanism of nucleotide incorporation opposite a cis-syn thymine-thymine (TT) dimer and an identical nondamaged sequence by yeast Polν. Polη displayed "burst" kinetics for nucleotide incorporation opposite both the damaged and nondamaged templates. Although a slight decrease occurred in the affinity (Kd) of nucleotide binding opposite the TT dimer relative to the nondamaged template, the rate (kpol) of nucleotide incorporation was the same whether the template was damaged or nondamaged. These results strongly support a mechanism in which the nucleotide is directly inserted opposite the TT dimer by using its intrinsic base-pairing ability without any hindrance from the distorted geometry of the lesion.

Original languageEnglish (US)
Pages (from-to)12093-12098
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume100
Issue number21
DOIs
StatePublished - Oct 14 2003

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Pyrimidine Dimers
Thymine
DNA-Directed DNA Polymerase
Nucleotides
Yeasts
Ultraviolet Rays
Base Pairing

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

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abstract = "DNA polymerase η (Polη) has the unique ability to replicate through UV-light-induced cyclobutane pyrimidine dimers. Here we use pre-steady-state kinetic analyses to examine the mechanism of nucleotide incorporation opposite a cis-syn thymine-thymine (TT) dimer and an identical nondamaged sequence by yeast Polν. Polη displayed {"}burst{"} kinetics for nucleotide incorporation opposite both the damaged and nondamaged templates. Although a slight decrease occurred in the affinity (Kd) of nucleotide binding opposite the TT dimer relative to the nondamaged template, the rate (kpol) of nucleotide incorporation was the same whether the template was damaged or nondamaged. These results strongly support a mechanism in which the nucleotide is directly inserted opposite the TT dimer by using its intrinsic base-pairing ability without any hindrance from the distorted geometry of the lesion.",
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AU - Prakash, Louise

AU - Prakash, Satya

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N2 - DNA polymerase η (Polη) has the unique ability to replicate through UV-light-induced cyclobutane pyrimidine dimers. Here we use pre-steady-state kinetic analyses to examine the mechanism of nucleotide incorporation opposite a cis-syn thymine-thymine (TT) dimer and an identical nondamaged sequence by yeast Polν. Polη displayed "burst" kinetics for nucleotide incorporation opposite both the damaged and nondamaged templates. Although a slight decrease occurred in the affinity (Kd) of nucleotide binding opposite the TT dimer relative to the nondamaged template, the rate (kpol) of nucleotide incorporation was the same whether the template was damaged or nondamaged. These results strongly support a mechanism in which the nucleotide is directly inserted opposite the TT dimer by using its intrinsic base-pairing ability without any hindrance from the distorted geometry of the lesion.

AB - DNA polymerase η (Polη) has the unique ability to replicate through UV-light-induced cyclobutane pyrimidine dimers. Here we use pre-steady-state kinetic analyses to examine the mechanism of nucleotide incorporation opposite a cis-syn thymine-thymine (TT) dimer and an identical nondamaged sequence by yeast Polν. Polη displayed "burst" kinetics for nucleotide incorporation opposite both the damaged and nondamaged templates. Although a slight decrease occurred in the affinity (Kd) of nucleotide binding opposite the TT dimer relative to the nondamaged template, the rate (kpol) of nucleotide incorporation was the same whether the template was damaged or nondamaged. These results strongly support a mechanism in which the nucleotide is directly inserted opposite the TT dimer by using its intrinsic base-pairing ability without any hindrance from the distorted geometry of the lesion.

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