The molecular architecture of the mammalian DNA repair enzyme, polynucleotide kinase.
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.
Bernstein
Nina K
NK
Department of Biochemistry, 4-74 Medical Sciences Building, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
Williams
R Scott
RS
Rakovszky
Melissa L
ML
Cui
Diana
D
Green
Ruth
R
Karimi-Busheri
Feridoun
F
Mani
Rajam S
RS
Galicia
Sarah
S
Koch
C Anne
CA
Cass
Carol E
CE
Durocher
Daniel
D
Weinfeld
Michael
M
Glover
J N Mark
JN
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
D018076:DNA, Complementary; D009841:Oligonucleotides; D005982:Glutathione Transferase; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D010744:Phosphoric Monoester Hydrolases; D014357:Trypsin