The Molecular Architecture of the Mammalian DNA Repair Enzyme, Polynucleotide Kinase

• Published in: Molecular cell Vol. 17; no. 5; pp. 657 - 670
• Main Authors: Bernstein, Nina K., Williams, R. Scott, Rakovszky, Melissa L., Cui, Diana, Green, Ruth, Karimi-Busheri, Feridoun, Mani, Rajam S., Galicia, Sarah, Koch, C. Anne, Cass, Carol E., Durocher, Daniel, Weinfeld, Michael, Glover, J.N. Mark
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 04.03.2005
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Animals; Binding Sites; Catalytic Domain; Cloning, Molecular; Crystallography, X-Ray; DNA Repair; DNA, Complementary - metabolism; Dose-Response Relationship, Drug; Glutathione Transferase - metabolism; Kinetics; Mice; Models, Molecular; Molecular Sequence Data; Oligonucleotides - chemistry; Phosphoric Monoester Hydrolases - chemistry; Phosphorylation; Polynucleotide 5'-Hydroxyl-Kinase - chemistry; Polynucleotide 5'-Hydroxyl-Kinase - physiology; Protein Binding; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Sequence Homology, Amino Acid; Spectrometry, Fluorescence; Static Electricity; Substrate Specificity; Trypsin - chemistry
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2005.02.012

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.