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

Department of Biochemistry, 4-74 Medical Sciences Building, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
Molecular Cell (Impact Factor: 14.02). 04/2005; 17(5):657-70. DOI: 10.1016/j.molcel.2005.02.012
Source: PubMed


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.


Available from: Michael Weinfeld
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    • "Mj) (pdb code: 3am1, Z = 9.2, rmsd = 3.2). All eight proteins have a P-loop, a common motif 160 GXXXXGKT 167 in ATP-and GTP-binding proteins and have been assigned as kinases or phosphotransferases (Bernstein et al., 2005; Galburt et al., 2002; Khoo et al., 2007; Mutschler et al., 2011; Sherrer et al., 2011; Wang et al., 2002; Yuen et al., 1999). 3D alignment of eight kinase structures and AvrRxo1 revealed that they all have common fold core structures of the N-terminal kinase domain of T4pnk, which are structurally conserved in spite of very low pair wise sequence identity. "
    Dataset: mmc1

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    • "The atomistic model of flexibly tethered PNKP was built based on the PNKP crystal structure [104], SAXS models of PNKP [105] and full length XRCC4 (blue) [74]. The model shows a far-reaching enzyme in the process of interacting with a DSB. "
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    ABSTRACT: Non-homologous end joining (NHEJ) is the major pathway for repair of DNA double-strand breaks (DSBs) in human cells. NHEJ is also needed for V(D)J recombination and the development of T and B cells in vertebrate immune systems, and acts in both the generation and prevention of non-homologous chromosomal translocations, a hallmark of genomic instability and many human cancers. X-ray crystal structures, cryo-electron microscopy envelopes, and small angle X-ray scattering (SAXS) solution conformations and assemblies are defining most of the core protein components for NHEJ: Ku70/Ku80 heterodimer; the DNA dependent protein kinase catalytic subunit (DNA-PKcs); the structure-specific endonuclease Artemis along with polynucleotide kinase/phosphatase (PNKP), aprataxin and PNKP related protein (APLF); the scaffolding proteins XRCC4 and XLF (XRCC4-like factor); DNA polymerases, and DNA ligase IV (Lig IV). The dynamic assembly of multi-protein NHEJ complexes at DSBs is regulated in part by protein phosphorylation. The basic steps of NHEJ have been biochemically defined to require: (1) DSB detection by the Ku heterodimer with subsequent DNA-PKcs tethering to form the DNA-PKcs-Ku-DNA complex (termed DNA-PK), (2) lesion processing, and (3) DNA end ligation by Lig IV, which functions in complex with XRCC4 and XLF. The current integration of structures by combined methods is resolving puzzles regarding the mechanisms, coordination and regulation of these three basic steps. Overall, structural results suggest the NHEJ system forms a flexing scaffold with the DNA-PKcs HEAT repeats acting as compressible macromolecular springs suitable to store and release conformational energy to apply forces to regulate NHEJ complexes and the DNA substrate for DNA end protection, processing, and ligation.
    DNA repair 05/2014; 17. DOI:10.1016/j.dnarep.2014.02.009 · 3.11 Impact Factor
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    • "We noted that whereas PNKPE326K, PNKPT424Gfs48X and PNKPexon15Δfs4X exhibited similar chromatographic properties to WT PNKP during purification, PNKPL176F failed to bind a cation exchange column (Supplementary Figure S1). As a negative control for DNA phosphatase activity, we also expressed and purified in parallel the protein PNKPD171N, which harbours a mutation in the DNA phosphatase catalytic domain and retains little or no DNA phosphatase activity (15,22,23). "
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    ABSTRACT: Microcephaly with early-onset, intractable seizures and developmental delay (MCSZ) is a hereditary disease caused by mutations in polynucleotide kinase/phosphatase (PNKP), a DNA strand break repair protein with DNA 5′-kinase and DNA 3′-phosphatase activity. To investigate the molecular basis of this disease, we examined the impact of MCSZ mutations on PNKP activity in vitro and in cells. Three of the four mutations currently associated with MCSZ greatly reduce or ablate DNA kinase activity of recombinant PNKP at 30°C (L176F, T424Gfs48X and exon15Δfs4X), but only one of these mutations reduces DNA phosphatase activity under the same conditions (L176F). The fourth mutation (E326K) has little impact on either DNA kinase or DNA phosphatase activity at 30°C, but is less stable than the wild-type enzyme at physiological temperature. Critically, all of the MCSZ mutations identified to date result in ∼10-fold reduced cellular levels of PNKP protein, and reduced rates of chromosomal DNA strand break repair. Together, these data suggest that all four known MCSZ mutations reduce the cellular stability and level of PNKP protein, with three mutations likely ablating cellular DNA 5′-kinase activity and all of the mutations greatly reducing cellular DNA 3′-phosphatase activity.
    Nucleic Acids Research 04/2012; 40(14):6608-19. DOI:10.1093/nar/gks318 · 9.11 Impact Factor
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