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Proc Natl Acad Sci U S A. 2011 Dec 27;108(52):21022-7. doi: 10.1073/pnas.1112036108. Epub 2011 Dec 14.

Structural basis for the phosphatase activity of polynucleotide kinase/phosphatase on single- and double-stranded DNA substrates.

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1
Department of Biochemistry, University of Alberta, Edmonton, AB, Canada T6G 2H7.

Abstract

Polynucleotide kinase/phosphatase (PNKP) is a critical mammalian DNA repair enzyme that generates 5'-phosphate and 3'-hydroxyl groups at damaged DNA termini that are required for subsequent processing by DNA ligases and polymerases. The PNKP phosphatase domain recognizes 3'-phosphate termini within DNA nicks, gaps, or at double- or single-strand breaks. Here we present a mechanistic rationale for the recognition of damaged DNA termini by the PNKP phosphatase domain. The crystal structures of PNKP bound to single-stranded DNA substrates reveals a narrow active site cleft that accommodates a single-stranded substrate in a sequence-independent manner. Biochemical studies suggest that the terminal base pairs of double-stranded substrates near the 3'-phosphate are destabilized by PNKP to allow substrate access to the active site. A positively charged surface distinct from the active site specifically facilitates interactions with double-stranded substrates, providing a complex DNA binding surface that enables the recognition of diverse substrates.

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PMID:
22171004
PMCID:
PMC3248541
DOI:
10.1073/pnas.1112036108
[Indexed for MEDLINE]
Free PMC Article
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