Nucleic Acids Research Advance Access originally published online on August 11, 2009
Nucleic Acids Research 2009 37(18):6161-6173; doi:10.1093/nar/gkp597
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Nucleic Acids Research, 2009, Vol. 37, No. 18 6161-6173
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acid Enzymes |
Mechanism of DNA substrate recognition by the mammalian DNA repair enzyme, Polynucleotide Kinase
1Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada, 2Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA, 3Department of Experimental Oncology, Cross Cancer Institute, Edmonton, Alberta, Canada, 4Department of Mathematics and Computer Science, University of Missouri in St. Louis, St. Louis, MO 63121, 5Department of Molecular Biology, Skaggs Institute of Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037 and 6Life Sciences Division, Department of Molecular Biology, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA
*To whom correspondence should be addressed. Tel: +1 780 492 2136; Fax: +1 780 492 0886; Email: mark.glover{at}ualberta.ca
Received April 7, 2009. Revised June 29, 2009. Accepted June 30, 2009.
Mammalian polynucleotide kinase (mPNK) is a critical DNA repair enzyme whose 5'-kinase and 3'-phoshatase activities function with poorly understood but striking specificity to restore 5'-phosphate/3'-hydroxyl termini at sites of DNA damage. Here we integrated site-directed mutagenesis and small-angle X-ray scattering (SAXS) combined with advanced computational approaches to characterize the conformational variability and DNA-binding properties of mPNK. The flexible attachment of the FHA domain to the catalytic segment, elucidated by SAXS, enables the interactions of mPNK with diverse DNA substrates and protein partners required for effective orchestration of DNA end repair. Point mutations surrounding the kinase active site identified two substrate recognition surfaces positioned to contact distinct regions on either side of the phosphorylated 5'-hydroxyl. DNA substrates bind across the kinase active site cleft to position the double-stranded portion upstream of the 5'-hydroxyl on one side, and the 3'-overhang on the opposite side. The bipartite DNA-binding surface of the mPNK kinase domain explains its preference for recessed 5'-termini, structures that would be encountered in the course of DNA strand break repair.
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