title: The molecular architecture of the mammalian DNA repair enzyme, polynucleotide kinase.
abstract: Mammalian polynucleotide kinase (PNK) is a key component of both the base excision repair (BER) and nonhomologous end-joining (NHEJ) DNA repair pathways. PNK acts as a 5'-kinase/3'-phosphatase to create 5'-phosphate/3'-hydroxyl termini, which are a necessary prerequisite for ligation during repair. PNK is recruited to repair complexes through interactions between its N-terminal FHA domain and phosphorylated components of either pathway. Here, we describe the crystal structure of intact mammalian PNK and a structure of the PNK FHA bound to a cognate phosphopeptide. The kinase domain has a broad substrate binding pocket, which preferentially recognizes double-stranded substrates with recessed 5' termini. In contrast, the phosphatase domain efficiently dephosphorylates single-stranded 3'-phospho termini as well as double-stranded substrates. The FHA domain is linked to the kinase/phosphatase catalytic domain by a flexible tether, and it exhibits a mode of target selection based on electrostatic complementarity between the binding surface and the phosphothreonine peptide.
authors.affiliation: Department of Biochemistry, 4-74 Medical Sciences Building, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors.forename: Nina K
authors.initials: NK
authors.lastname: Bernstein
authors.forename: R Scott
authors.initials: RS
authors.lastname: Williams
authors.forename: Melissa L
authors.initials: ML
authors.lastname: Rakovszky
authors.forename: Diana
authors.initials: D
authors.lastname: Cui
authors.forename: Ruth
authors.initials: R
authors.lastname: Green
authors.forename: Feridoun
authors.initials: F
authors.lastname: Karimi-Busheri
authors.forename: Rajam S
authors.initials: RS
authors.lastname: Mani
authors.forename: Sarah
authors.initials: S
authors.lastname: Galicia
authors.forename: C Anne
authors.initials: CA
authors.lastname: Koch
authors.forename: Carol E
authors.initials: CE
authors.lastname: Cass
authors.forename: Daniel
authors.initials: D
authors.lastname: Durocher
authors.forename: Michael
authors.initials: M
authors.lastname: Weinfeld
authors.forename: J N Mark
authors.initials: JN
authors.lastname: Glover
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004260:DNA Repair; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005982:Glutathione Transferase / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry; D010766:Phosphorylation; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000737:chemistry / Q000502:physiology*; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D055672:Static Electricity; D013379:Substrate Specificity; D014357:Trypsin / Q000737:chemistry
chemical_list: D018076:DNA, Complementary; D009841:Oligonucleotides; D005982:Glutathione Transferase; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D010744:Phosphoric Monoester Hydrolases; D014357:Trypsin