RESUMO
Cancers with hereditary defects in homologous recombination rely on DNA polymerase θ (pol θ) for repair of DNA double-strand breaks. During end joining, pol θ aligns microhomology tracts internal to 5'-resected broken ends. An unidentified nuclease trims the 3' ends before synthesis can occur. Here we report that a nuclease activity, which differs from the proofreading activity often associated with DNA polymerases, is intrinsic to the polymerase domain of pol θ. Like the DNA synthesis activity, the nuclease activity requires conserved metal-binding residues, metal ions, and dNTPs and is inhibited by ddNTPs or chain-terminated DNA. Our data indicate that pol θ repurposes metal ions in the polymerase active site for endonucleolytic cleavage and that the polymerase-active and end-trimming conformations of the enzyme are distinct. We reveal a nimble strategy of substrate processing that allows pol θ to trim or extend DNA depending on the DNA repair context.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA/metabolismo , Endonucleases/metabolismo , Metais/metabolismo , Linhagem Celular , DNA/genética , DNA Polimerase Dirigida por DNA/genética , Endonucleases/genética , Humanos , DNA Polimerase tetaRESUMO
In this issue of Genes & Development, Gong and colleagues (pp. 46-58) bring to light a functional role for plant homeodomain finger 11 (PHF11) in 5' end resection at DNA double-strand breaks (DSBs). Using the proteomics of isolated chromatin segments (PICh) technique to purify deprotected telomeres, PHF11 was enriched as cells mounted a DNA damage response (DDR) against exposed chromosome ends. The study reveals interactions between PHF11 and multiple DNA repair proteins and suggests that PHF11 mediates 5' end resection by negotiating RPA-coated DNA repair intermediates. This finding provides a novel context for mediator-catalyzed RPA exchanges during the multistep process of homologous recombination (HR).
Assuntos
Reparo do DNA , Exodesoxirribonucleases/genética , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/genética , Recombinação HomólogaRESUMO
Base excision repair (BER) is the main pathway protecting cells from the continuous damage to DNA inflicted by reactive oxygen species. BER is initiated by DNA glycosylases, each of which repairs a particular class of base damage. NTHL1, a bifunctional DNA glycosylase, possesses both glycolytic and ß-lytic activities with a preference for oxidized pyrimidine substrates. Defects in human NTHL1 drive a class of polyposis colorectal cancer. We report the first X-ray crystal structure of hNTHL1, revealing an open conformation not previously observed in the bacterial orthologs. In this conformation, the six-helical barrel domain comprising the helix-hairpin-helix (HhH) DNA binding motif is tipped away from the iron sulphur cluster-containing domain, requiring a conformational change to assemble a catalytic site upon DNA binding. We found that the flexibility of hNTHL1 and its ability to adopt an open configuration can be attributed to an interdomain linker. Swapping the human linker sequence for that of Escherichia coli yielded a protein chimera that crystallized in a closed conformation and had a reduced activity on lesion-containing DNA. This large scale interdomain rearrangement during catalysis is unprecedented for a HhH superfamily DNA glycosylase and provides important insight into the molecular mechanism of hNTHL1.
Assuntos
Domínio Catalítico , Reparo do DNA , DNA/química , Desoxirribonuclease (Dímero de Pirimidina)/química , Domínios Proteicos , Sequência de Aminoácidos , Biocatálise , DNA/genética , DNA/metabolismo , Desoxirribonuclease (Dímero de Pirimidina)/genética , Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , Humanos , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Pirimidinas/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Phosphonoformic acid (PFA, foscarnet) belongs to a class of antiviral drugs that inhibit the human cytomegalovirus DNA polymerase (UL54) by mimicking the pyrophosphate leaving group of the nucleotide transfer reaction. Difficulties expressing UL54 have hampered investigation of the precise structural requirements rendering inhibition by this drug. However, a previously engineered chimeric DNA polymerase, constructed by mutating the homologous polymerase from bacteriophage RB69 (gp43) to express several variable elements from UL54, can bypass this obstacle because of its favorable expression and acquired sensitivity to PFA (Tchesnokov, E. P., Obikhod, A., Schinazi, R. F., and Götte, M. (2008) J. Biol. Chem. 283, 34218-34228). Here, we compare two crystal structures that depict the chimeric DNA polymerase with and without PFA bound. PFA is visualized for the first time in the active site of a DNA polymerase, where interactions are resolved between the PP(i) mimic and two basic residues absolutely conserved in the fingers domain of family B polymerases. PFA also chelates metal ion B, the cation that contacts the triphosphate tail of the incoming nucleotide. These DNA complexes utilize a primer-template pair enzymatically chain-terminated by incorporation of acyclo-GMP, the phosphorylated form of the anti-herpes drug acyclovir. We postulate that the V478W mutation present in the chimera is critical in that it pushes the fingers domain to more readily adopt the closed conformation whether or not the drug is bound. The closed state of the fingers domain traps the variant polymerase in the untranslocated state and increases affinity for PFA. This finding provides a model for the mechanism of UL54 stalling by PFA.
Assuntos
Citomegalovirus/enzimologia , DNA Polimerase Dirigida por DNA/química , Foscarnet/química , Modelos Moleculares , Proteínas Virais/química , Aciclovir/análogos & derivados , Aciclovir/química , Substituição de Aminoácidos , Cristalografia por Raios X , Citomegalovirus/genética , DNA Polimerase Dirigida por DNA/genética , Humanos , Mutação Puntual , Estrutura Terciária de Proteína , Proteínas Virais/genéticaRESUMO
DNA polymerase theta (Polθ) is an attractive synthetic lethal target for drug discovery, predicted to be efficacious against breast and ovarian cancers harboring BRCA-mutant alleles. Here, we describe our hit-to-lead efforts in search of a selective inhibitor of human Polθ (encoded by POLQ). A high-throughput screening campaign of 350,000 compounds identified an 11 micromolar hit, giving rise to the N2-substituted fused pyrazolo series, which was validated by biophysical methods. Structure-based drug design efforts along with optimization of cellular potency and ADME ultimately led to the identification of RP-6685: a potent, selective, and orally bioavailable Polθ inhibitor that showed in vivo efficacy in an HCT116 BRCA2-/- mouse tumor xenograft model.
Assuntos
DNA Polimerase Dirigida por DNA , Neoplasias Ovarianas , Animais , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Desenho de Fármacos , Descoberta de Drogas , Feminino , Humanos , CamundongosRESUMO
5-Hydroxycytosine (5-OHC) is a stable oxidation product of cytosine associated with an increased frequency of C â T transition mutations. When this lesion escapes recognition by the base excision repair pathway and persists to serve as a templating base during DNA synthesis, replicative DNA polymerases often misincorporate dAMP at the primer terminus, which can lead to fixation of mutations and subsequent disease. To characterize the dynamics of DNA synthesis opposite 5-OHC, we initiated a comparison of unmodified dCMP to 5-OHC, 5-fluorocytosine (5-FC), and 5-methylcytosine (5-MEC) in which these bases act as templates in the active site of RB69 gp43, a high-fidelity DNA polymerase sharing homology with human replicative DNA polymerases. This study presents the first crystal structure of any DNA polymerase binding this physiologically important premutagenic DNA lesion, showing that while dGMP is stabilized by 5-OHC through normal Watson-Crick base pairing, incorporation of dAMP leads to unstacking and instability in the template. Furthermore, the electronegativity of the C5 substituent appears to be important in the miscoding potential of these cytosine-like templates. While dAMP is incorporated opposite 5-OHC ~5 times more efficiently than opposite unmodified dCMP, an elevated level of incorporation is also observed opposite 5-FC but not 5-MEC. Taken together, these data imply that the nonuniform templating by 5-OHC is due to weakened stacking capabilities, which allows dAMP incorporation to proceed in a manner similar to that observed opposite abasic sites.
Assuntos
Bacteriófagos/enzimologia , Citosina/análogos & derivados , Replicação do DNA , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Bacteriófagos/química , Pareamento de Bases , Domínio Catalítico , Citosina/química , Citosina/metabolismo , DNA Polimerase Dirigida por DNA/genética , Moldes Genéticos , Proteínas Virais/genéticaRESUMO
The POLQ gene encodes DNA polymerase θ, a 2590 amino acid protein product harboring DNA-dependent ATPase, template-dependent DNA polymerase, dNTP-dependent endonuclease, and 5'-dRP lyase functions. Polymerase θ participates at an essential step of a DNA double-strand break repair pathway able to join 5'-resected substrates by locating and pairing microhomologies present in 3'-overhanging single-stranded tails, cleaving the extraneous 3'-DNA by dNTP-dependent end-processing, before extending the nascent 3' end from the microhomology annealing site. Metazoans require polymerase θ for full resistance to DNA double-strand break inducing agents but can survive knockout of the POLQ gene. Cancer cells with compromised homologous recombination, or other DNA repair defects, over-utilize end-joining by polymerase θ and often over-express the POLQ gene. This dependency points to polymerase θ as an ideal drug target candidate and multiple drug-development programs are now preparing to enter clinical trials with small-molecule inhibitors. Specific inhibitors of polymerase θ would not only be predicted to treat BRCA-mutant cancers, but could thwart accumulated resistance to current standard-of-care cancer therapies and overcome PARP-inhibitor resistance in patients. This article will discuss synthetic lethal strategies targeting polymerase θ in DNA damage-response-deficient cancers and summarize data, describing molecular structures and enzymatic functions.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , DNA Polimerase Dirigida por DNA/metabolismo , Neoplasias/tratamento farmacológico , Inibidores da Síntese de Ácido Nucleico/uso terapêutico , Animais , DNA Polimerase Dirigida por DNA/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Modelos Animais , Neoplasias/genética , Inibidores da Síntese de Ácido Nucleico/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Mutações Sintéticas Letais/efeitos dos fármacos , DNA Polimerase tetaRESUMO
DNA polymerase θ protects against genomic instability via an alternative end-joining repair pathway for DNA double-strand breaks. Polymerase θ is overexpressed in breast, lung and oral cancers, and reduction of its activity in mammalian cells increases sensitivity to double-strand break-inducing agents, including ionizing radiation. Reported here are crystal structures of the C-terminal polymerase domain from human polymerase θ, illustrating two potential modes of dimerization. One structure depicts insertion of ddATP opposite an abasic-site analog during translesion DNA synthesis. The second structure describes a cognate ddGTP complex. Polymerase θ uses a specialized thumb subdomain to establish unique upstream contacts to the primer DNA strand, including an interaction with the 3'-terminal phosphate from one of five distinctive insertion loops. These observations demonstrate how polymerase θ grasps the primer to bypass DNA lesions or extend poorly annealed DNA termini to mediate end-joining.
Assuntos
DNA Polimerase Dirigida por DNA/fisiologia , Sequência de Aminoácidos , Cristalografia por Raios X , DNA Polimerase Dirigida por DNA/química , Dimerização , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Alinhamento de Sequência , DNA Polimerase tetaRESUMO
THREE DNA POLYMERASES OF THE B FAMILY FUNCTION AT THE REPLICATION FORK IN EUKARYOTIC CELLS: DNA polymerases α, δ, and ε. DNA polymerase α, an heterotetramer composed of two primase subunits and two polymerase subunits, initiates replication. DNA polymerases δ and ε elongate the primers generated by pol α. The DNA polymerase from bacteriophage RB69 has served as a model for eukaryotic B family polymerases for some time. The recent crystal structures of pol δ, α, and ε revealed similarities but also a number of unexpected differences between the eukaryotic polymerases and their bacteriophage counterpart, and also among the three yeast polymerases. This review will focus on their shared structural elements as well as the features that are unique to each of these polymerases.
RESUMO
Deducing the structure of the DNA double helix in 1953 implied the mode of its replication: Watson-Crick (WC) base pairing might instruct an enzyme, now known as the DNA polymerase, during the synthesis of a daughter stand complementary to a single strand of the parental double helix. What has become increasingly clear in the last 60 years, however, is that adducted and oxidatively generated DNA bases are ubiquitous in physiological DNA, and all organisms conserve multiple DNA polymerases specialized for DNA synthesis opposite these damaged templates. Here, we review recent crystal structures depicting replicative and bypass DNA polymerases encountering two typical lesions arising from the oxidation of DNA: abasic sites, which block the replication fork, and the miscoding premutagenic lesion 7,8-dihydro-8-oxoguanine (8-oxoG).
Assuntos
Replicação do DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Animais , Pareamento de Bases/genética , Dano ao DNA , DNA Polimerase Dirigida por DNA/química , Desoxicitidina Monofosfato/metabolismo , Guanina/análogos & derivados , Guanina/metabolismo , Humanos , Modelos Moleculares , Nucleotídeos/metabolismo , Oxirredução , Moldes GenéticosRESUMO
Damage to DNA involving excision of the nucleobase at the N-glycosidic bond forms abasic sites. If a nucleotide becomes incorporated opposite an unrepaired abasic site during DNA synthesis, most B family polymerases obey the A-rule and preferentially incorporate dAMP without instruction from the template. In addition to being potentially mutagenic, abasic sites provide strong blocks to DNA synthesis. A previous crystal structure of an exonuclease deficient variant of the replicative B family DNA polymerase from bacteriophage RB69 (RB69 gp43 exo-) illustrated these properties, showing that the polymerase failed to translocate the DNA following insertion of dAMP opposite an abasic site. We examine four new structures depicting several steps of translesion DNA synthesis by RB69 gp43 exo-, employing a non-natural purine triphosphate analogue, 5-nitro-1-indolyl-2'-deoxyriboside-5'-triphosphate (5-NITP), that is incorporated more efficiently than dAMP opposite abasic sites. Our structures indicate that a dipole-induced dipole stacking interaction between the 5-nitro group and base 3' to the templating lesion explains the enhanced kinetics of 5-NITP. As with dAMP, the DNA fails to translocate following insertion of 5-NIMP, although distortions at the nascent primer terminus contribute less than previously thought in inducing the stall, given that 5-NIMP preserves relatively undistorted geometry at the insertion site following phosphoryl transfer. An open ternary configuration, novel in B family polymerases, reveals an initial template independent binding of 5-NITP adjacent to the active site of the open polymerase, suggesting that closure of the fingers domain shuttles the nucleotide to the active site while testing the substrate against the template.