title: Characterization of DNA Substrate Binding to the Phosphatase Domain of the DNA Repair Enzyme Polynucleotide Kinase/Phosphatase.
abstract: Polynucleotide kinase/phosphatase (PNKP) is a DNA strand break repair enzyme that uses separate 5' kinase and 3' phosphatase active sites to convert damaged 5'-hydroxyl/3'-phosphate strand termini to ligatable 5'-phosphate/3'-hydroxyl ends. The phosphatase active site has received particular attention as a target of inhibition in cancer therapy development. The phosphatase domain dephosphorylates a range of single- and double-stranded substrates; however, structural studies have shown that the phosphatase catalytic cleft can bind only single-stranded substrates. Here we use a catalytically inactive but structurally intact phosphatase mutant to probe interactions between PNKP and a variety of single- and double-stranded DNA substrates using an electrophoretic mobility shift assay. This work indicates that the phosphatase domain binds 3'-phosphorylated single-stranded DNAs in a manner that is highly dependent on the presence of the 3'-phosphate. Double-stranded substrate binding, in contrast, is not as dependent on the 3'-phosphate. Experiments comparing blunt-end, 3'-overhanging, and frayed-end substrates indicate that the predicted loss of energy due to base pair disruption upon binding of the phosphatase active site is likely balanced by favorable interactions between the liberated complementary strand and PNKP. Comparison of the effects on substrate binding of mutations within the phosphatase active site cleft with mutations in surrounding positively charged surfaces suggests that the surrounding surfaces are important for binding to double-stranded substrates. We further show that while fluorescence polarization methods can detect specific binding of single-stranded DNAs with the phosphatase domain, this method does not detect specific interactions between the PNKP phosphatase and double-stranded substrates.
authors.affiliation: Department of Biochemistry, University of Alberta , Edmonton, Alberta T6G 2H7, Canada.
authors.forename: Zahra
authors.initials: Z
authors.lastname: Havali-Shahriari
authors.affiliation: Department of Oncology, University of Alberta, Cross Cancer Institute , 11560 University Avenue, Edmonton, Alberta T6G 1Z2, Canada.
authors.forename: Michael
authors.initials: M
authors.lastname: Weinfeld
authors.affiliation: Department of Biochemistry, University of Alberta , Edmonton, Alberta T6G 2H7, Canada.
authors.forename: J N Mark
authors.identifier: 0000-0003-2733-7052
authors.initials: JN
authors.lastname: Glover
mesh_terms: D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D051379:Mice; D062105:Molecular Docking Simulation; D009154:Mutation; D010710:Phosphates / Q000737:chemistry* / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D004277:DNA, Single-Stranded; D010710:Phosphates; D011994:Recombinant Proteins; D004247:DNA; D017853:Phosphotransferases (Alcohol Group Acceptor); C000619445:Pnkp protein, mouse