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Nucleic Acids Research 2006 34(8):2230-2237; doi:10.1093/nar/gkl275
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Published online 28 April 2006

© The Author 2006. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org

Article

The phosphatase activity of mammalian polynucleotide kinase takes precedence over its kinase activity in repair of single strand breaks

Caroline J. Dobson and Sarah L. Allinson*

Biomedical Sciences Unit, Department of Biological Sciences, Lancaster University Lancaster, LA1 4YQ, UK

*To whom correspondence should be addressed. Tel: +44 1524 593 922; Fax: +44 1524 593 192; Email: s.allinson{at}lancaster.ac.uk

Received February 4, 2006. Accepted April 3, 2006.

The dual function mammalian DNA repair enzyme, polynucleotide kinase (PNK), facilitates strand break repair through catalysis of 5'-hydroxyl phosphorylation and 3'-phosphate dephosphorylation. We have examined the relative activities of the kinase and phosphatase functions of PNK using a novel assay, which allows the simultaneous characterization of both activities in processing nicks and gaps containing both 3'-phosphate and 5'-hydroxyl. Under multiple turnover conditions the phosphatase activity of the purified enzyme is significantly more active than its kinase activity. Consistent with this result, phosphorylation of the 5'-hydroxyl is rate limiting in cell extract mediated-repair of a nicked substrate. On characterizing the effects of individually mutating the two active sites of PNK we find that while site-directed mutagenesis of the kinase domain of PNK does not affect its phosphatase activity, disruption of the phosphatase domain also abrogates kinase function. This loss of kinase function requires the presence of a 3'-phosphate, but it need not be present in the same strand break as the 5'-hydroxyl. PNK preferentially binds 3'-phosphorylated substrates and DNA binding to the phosphatase domain blocks further DNA binding by the kinase domain.


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[Abstract] [Full Text] [PDF]


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