title: Mechanism of DNA substrate recognition by the mammalian DNA repair enzyme, Polynucleotide Kinase.
abstract: 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.
authors: Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors: N K
authors: NK
authors: Bernstein
authors: M
authors: M
authors: Hammel
authors: R S
authors: RS
authors: Mani
authors: M
authors: M
authors: Weinfeld
authors: M
authors: M
authors: Pelikan
authors: J A
authors: JA
authors: Tainer
authors: J N M
authors: JN
authors: Glover
mesh_terms: D000818:Animals; D002384:Catalysis; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
chemical_list: D004247:DNA; D011116:Polynucleotide 5'-Hydroxyl-Kinase