Evidence for a Watson-Crick hydrogen bonding requirement in DNA synthesis by human DNA polymerase κ

William T. Wolfle, M. Todd Washington, Eric T. Kool, Thomas E. Spratt, Sandra A. Helquist, Louise Prakash, Satya Prakash

Research output: Contribution to journalArticle

49 Citations (Scopus)

Abstract

The efficiency and fidelity of nucleotide incorporation by high-fidelity replicative DNA polymerases (Pols) are governed by the geometric constraints imposed upon the nascent base pair by the active site. Consequently, these polymerases can efficiently and accurately replicate through the template bases which are isosteric to natural DNA bases but which lack the ability to engage in Watson-Crick (W-C) hydrogen bonding. DNA synthesis by Polη, a low-fidelity polymerase able to replicate through DNA lesions, however, is inhibited in the presence of such an analog, suggesting a dependence of this polymerase upon W-C hydrogen bonding. Here we examine whether human Polκ, which differs from Polη in having a higher fidelity and which, unlike polη, is inhibited at inserting nucleotides opposite DNA lesions, shows less of a dependence upon W-C hydrogen bonding than does Polη. We find that an isosteric thymidine analog is replicated with low efficiency by Polκ, whereas a nucleobase analog lacking minor-groove H bonding potential is replicated with high efficiency. These observations suggest that both Polη and Polκ rely on W-C hydrogen bonding for localizing the nascent base pair in the active site for the polymerization reaction to occur, thus overcoming these enzymes' low geometric selectivity.

Original languageEnglish (US)
Pages (from-to)7137-7143
Number of pages7
JournalMolecular and Cellular Biology
Volume25
Issue number16
DOIs
StatePublished - Aug 2005

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DNA-Directed DNA Polymerase
Hydrogen Bonding
DNA
Base Pairing
Catalytic Domain
Nucleotides
Polymerization
Thymidine
Enzymes

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this

Evidence for a Watson-Crick hydrogen bonding requirement in DNA synthesis by human DNA polymerase κ. / Wolfle, William T.; Washington, M. Todd; Kool, Eric T.; Spratt, Thomas E.; Helquist, Sandra A.; Prakash, Louise; Prakash, Satya.

In: Molecular and Cellular Biology, Vol. 25, No. 16, 08.2005, p. 7137-7143.

Research output: Contribution to journalArticle

Wolfle, William T. ; Washington, M. Todd ; Kool, Eric T. ; Spratt, Thomas E. ; Helquist, Sandra A. ; Prakash, Louise ; Prakash, Satya. / Evidence for a Watson-Crick hydrogen bonding requirement in DNA synthesis by human DNA polymerase κ. In: Molecular and Cellular Biology. 2005 ; Vol. 25, No. 16. pp. 7137-7143.
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