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1.
Annu Rev Biochem ; 91: 133-155, 2022 06 21.
Artículo en Inglés | MEDLINE | ID: mdl-35287470

RESUMEN

Our current view of how DNA-based genomes are efficiently and accurately replicated continues to evolve as new details emerge on the presence of ribonucleotides in DNA. Ribonucleotides are incorporated during eukaryotic DNA replication at rates that make them the most common noncanonical nucleotide placed into the nuclear genome, they are efficiently repaired, and their removal impacts genome integrity. This review focuses on three aspects of this subject: the incorporation of ribonucleotides into the eukaryotic nuclear genome during replication by B-family DNA replicases, how these ribonucleotides are removed, and the consequences of their presence or removal for genome stability and disease.


Asunto(s)
Replicación del ADN , Inestabilidad Genómica , Ribonucleótidos , ADN/genética , ADN/metabolismo , Reparación del ADN , Eucariontes/genética , Eucariontes/metabolismo , Nucleotidiltransferasas/genética , Ribonucleótidos/genética , Ribonucleótidos/metabolismo
2.
Nat Rev Mol Cell Biol ; 17(6): 350-63, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-27093943

RESUMEN

The information encoded in DNA is influenced by the presence of non-canonical nucleotides, the most frequent of which are ribonucleotides. In this Review, we discuss recent discoveries about ribonucleotide incorporation into DNA during replication by the three major eukaryotic replicases, DNA polymerases α, δ and ε. The presence of ribonucleotides in DNA causes short deletion mutations and may result in the generation of single- and double-strand DNA breaks, leading to genome instability. We describe how these ribonucleotides are removed from DNA through ribonucleotide excision repair and by topoisomerase I. We discuss the biological consequences and the physiological roles of ribonucleotides in DNA, and consider how deficiencies in their removal from DNA may be important in the aetiology of disease.


Asunto(s)
Replicación del ADN , Ribonucleótidos/metabolismo , Animales , ADN/biosíntesis , ADN/genética , Reparación del ADN , Humanos , Polimerizacion
3.
Cell ; 139(1): 87-99, 2009 Oct 02.
Artículo en Inglés | MEDLINE | ID: mdl-19804755

RESUMEN

The Nijmegen breakage syndrome 1 (Nbs1) subunit of the Mre11-Rad50-Nbs1 (MRN) complex protects genome integrity by coordinating double-strand break (DSB) repair and checkpoint signaling through undefined interactions with ATM, MDC1, and Sae2/Ctp1/CtIP. Here, fission yeast and human Nbs1 structures defined by X-ray crystallography and small angle X-ray scattering (SAXS) reveal Nbs1 cardinal features: fused, extended, FHA-BRCT(1)-BRCT(2) domains flexibly linked to C-terminal Mre11- and ATM-binding motifs. Genetic, biochemical, and structural analyses of an Nbs1-Ctp1 complex show Nbs1 recruits phosphorylated Ctp1 to DSBs via binding of the Nbs1 FHA domain to a Ctp1 pThr-Asp motif. Nbs1 structures further identify an extensive FHA-BRCT interface, a bipartite MDC1-binding scaffold, an extended conformational switch, and the molecular consequences associated with cancer predisposing Nijmegen breakage syndrome mutations. Tethering of Ctp1 to a flexible Nbs1 arm suggests a mechanism for restricting DNA end processing and homologous recombination activities of Sae2/Ctp1/CtIP to the immediate vicinity of DSBs.


Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas Cromosómicas no Histona/química , Reparación del ADN , Proteínas Nucleares/química , Proteínas de Schizosaccharomyces pombe/química , Ácido Anhídrido Hidrolasas , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Cristalografía por Rayos X , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Humanos , Proteína Homóloga de MRE11 , Modelos Moleculares , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estructura Terciaria de Proteína , Dispersión del Ángulo Pequeño , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo
4.
Crit Rev Biochem Mol Biol ; 56(1): 109-124, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33461360

RESUMEN

Ribonucleotides are the most abundant non-canonical nucleotides in the genome. Their vast presence and influence over genome biology is becoming increasingly appreciated. Here we review the recent progress made in understanding their genomic presence, incorporation characteristics and usefulness as biomarkers for polymerase enzymology. We also discuss ribonucleotide processing, the genetic consequences of unrepaired ribonucleotides in DNA and evidence supporting the significance of their transient presence in the nuclear genome.


Asunto(s)
Replicación del ADN/genética , ADN/genética , ADN/metabolismo , Ribonucleótidos/genética , Ribonucleótidos/metabolismo , Animales , Biomarcadores/metabolismo , Núcleo Celular/metabolismo , Reparación del ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Genoma Mitocondrial , Inestabilidad Genómica , Humanos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
5.
Cell ; 135(1): 97-109, 2008 Oct 03.
Artículo en Inglés | MEDLINE | ID: mdl-18854158

RESUMEN

Mre11 forms the core of the multifunctional Mre11-Rad50-Nbs1 (MRN) complex that detects DNA double-strand breaks (DSBs), activates the ATM checkpoint kinase, and initiates homologous recombination (HR) repair of DSBs. To define the roles of Mre11 in both DNA bridging and nucleolytic processing during initiation of DSB repair, we combined small-angle X-ray scattering (SAXS) and crystal structures of Pyrococcus furiosus Mre11 dimers bound to DNA with mutational analyses of fission yeast Mre11. The Mre11 dimer adopts a four-lobed U-shaped structure that is critical for proper MRN complex assembly and for binding and aligning DNA ends. Further, mutations blocking Mre11 endonuclease activity impair cell survival after DSB induction without compromising MRN complex assembly or Mre11-dependant recruitment of Ctp1, an HR factor, to DSBs. These results show how Mre11 dimerization and nuclease activities initiate repair of DSBs and collapsed replication forks, as well as provide a molecular foundation for understanding cancer-causing Mre11 mutations in ataxia telangiectasia-like disorder (ATLD).


Asunto(s)
Proteínas Arqueales/metabolismo , Reparación del ADN , ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Pyrococcus furiosus/química , Proteínas Arqueales/química , Proteínas Arqueales/genética , Cristalografía por Rayos X , ADN/química , Roturas del ADN de Doble Cadena , Análisis Mutacional de ADN , Dimerización , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/genética , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/genética , Modelos Moleculares , Dispersión del Ángulo Pequeño , Schizosaccharomyces/genética , Técnicas del Sistema de Dos Híbridos , Difracción de Rayos X
6.
EMBO J ; 36(3): 361-373, 2017 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-27932446

RESUMEN

Ribonuclease activity of topoisomerase I (Top1) causes DNA nicks bearing 2',3'-cyclic phosphates at ribonucleotide sites. Here, we provide genetic and biochemical evidence that DNA double-strand breaks (DSBs) can be directly generated by Top1 at sites of genomic ribonucleotides. We show that RNase H2-deficient yeast cells displayed elevated frequency of Rad52 foci, inactivation of RNase H2 and RAD52 led to synthetic lethality, and combined loss of RNase H2 and RAD51 induced slow growth and replication stress. Importantly, these phenotypes were rescued upon additional deletion of TOP1, implicating homologous recombination for the repair of Top1-induced damage at ribonuclelotide sites. We demonstrate biochemically that irreversible DSBs are generated by subsequent Top1 cleavage on the opposite strand from the Top1-induced DNA nicks at ribonucleotide sites. Analysis of Top1-linked DNA from pull-down experiments revealed that Top1 is covalently linked to the end of DNA in RNase H2-deficient yeast cells, supporting this model. Taken together, these results define Top1 as a source of DSBs and genome instability when ribonucleotides incorporated by the replicative polymerases are not removed by RNase H2.


Asunto(s)
Roturas del ADN de Doble Cadena , ADN-Topoisomerasas de Tipo I/metabolismo , ADN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Eliminación de Gen , Recombinasa Rad51/metabolismo , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Ribonucleasa H/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo
7.
Mol Cell ; 50(3): 437-43, 2013 May 09.
Artículo en Inglés | MEDLINE | ID: mdl-23603118

RESUMEN

To maintain genome stability, mismatch repair of nuclear DNA replication errors must be directed to the nascent strand, likely by DNA ends and PCNA. Here we show that the efficiency of mismatch repair in Saccharomyces cerevisiae is reduced by inactivating RNase H2, which nicks DNA containing ribonucleotides incorporated during replication. In strains encoding mutator polymerases, this reduction is preferential for repair of mismatches made by leading-strand DNA polymerase ε as compared to lagging-strand DNA polymerase δ. The results suggest that RNase-H2-dependent processing of ribonucleotides transiently present in DNA after replication may direct mismatch repair to the continuously replicated nascent leading strand.


Asunto(s)
Reparación de la Incompatibilidad de ADN , Replicación del ADN/genética , Ribonucleótidos/genética , Ribonucleótidos/metabolismo , ADN Polimerasa II/genética , ADN Polimerasa II/metabolismo , ADN Polimerasa III/genética , ADN Polimerasa III/metabolismo , Inestabilidad Genómica , Ribonucleasa H/genética , Ribonucleasa H/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
8.
Mol Cell ; 49(5): 1010-5, 2013 Mar 07.
Artículo en Inglés | MEDLINE | ID: mdl-23375499

RESUMEN

RNase H2-dependent ribonucleotide excision repair (RER) removes ribonucleotides incorporated during DNA replication. When RER is defective, ribonucleotides in the nascent leading strand of the yeast genome are associated with replication stress and genome instability. Here, we provide evidence that topoisomerase 1 (Top1) initiates an independent form of repair to remove ribonucleotides from genomic DNA. This Top1-dependent process activates the S phase checkpoint. Deleting TOP1 reverses this checkpoint activation and also relieves replication stress and genome instability in RER-defective cells. The results reveal an additional removal pathway for a very common lesion in DNA, and they imply that the "dirty" DNA ends created when Top1 incises ribonucleotides in DNA are responsible for the adverse consequences of ribonucleotides in RNase H2-defective cells.


Asunto(s)
Reparación del ADN , ADN-Topoisomerasas de Tipo I/metabolismo , ADN de Hongos/metabolismo , Ribonucleótidos/metabolismo , ADN-Topoisomerasas de Tipo I/genética , Inestabilidad Genómica , Ribonucleasa H/genética , Ribonucleasa H/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
9.
Nature ; 506(7486): 111-5, 2014 Feb 06.
Artículo en Inglés | MEDLINE | ID: mdl-24362567

RESUMEN

Faithful maintenance and propagation of eukaryotic genomes is ensured by three-step DNA ligation reactions used by ATP-dependent DNA ligases. Paradoxically, when DNA ligases encounter nicked DNA structures with abnormal DNA termini, DNA ligase catalytic activity can generate and/or exacerbate DNA damage through abortive ligation that produces chemically adducted, toxic 5'-adenylated (5'-AMP) DNA lesions. Aprataxin (APTX) reverses DNA adenylation but the context for deadenylation repair is unclear. Here we examine the importance of APTX to RNase-H2-dependent excision repair (RER) of a lesion that is very frequently introduced into DNA, a ribonucleotide. We show that ligases generate adenylated 5' ends containing a ribose characteristic of RNase H2 incision. APTX efficiently repairs adenylated RNA-DNA, and acting in an RNA-DNA damage response (RDDR), promotes cellular survival and prevents S-phase checkpoint activation in budding yeast undergoing RER. Structure-function studies of human APTX-RNA-DNA-AMP-Zn complexes define a mechanism for detecting and reversing adenylation at RNA-DNA junctions. This involves A-form RNA binding, proper protein folding and conformational changes, all of which are affected by heritable APTX mutations in ataxia with oculomotor apraxia 1. Together, these results indicate that accumulation of adenylated RNA-DNA may contribute to neurological disease.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Genoma Humano/genética , Proteínas Nucleares/metabolismo , ARN/metabolismo , Adenosina Monofosfato/metabolismo , Apraxias/genética , Ataxia Telangiectasia/genética , Supervivencia Celular , Ataxia Cerebelosa/congénito , ADN/química , Reparación del ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/metabolismo , Humanos , Hipoalbuminemia/genética , Modelos Moleculares , Mutación/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Conformación de Ácido Nucleico , Conformación Proteica , Pliegue de Proteína , ARN/química , Ribonucleasa H/metabolismo , Puntos de Control de la Fase S del Ciclo Celular , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Relación Estructura-Actividad , Zinc/metabolismo
10.
Crit Rev Biochem Mol Biol ; 51(1): 43-52, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26822554

RESUMEN

The eukaryotic nuclear genome is replicated asymmetrically, with the leading strand replicated continuously and the lagging strand replicated as discontinuous Okazaki fragments that are subsequently joined. Both strands are replicated with high fidelity, but the processes used to achieve high fidelity are likely to differ. Here we review recent studies of similarities and differences in the fidelity with which the three major eukaryotic replicases, DNA polymerases α, δ, and ɛ, replicate the leading and lagging strands with high nucleotide selectivity and efficient proofreading. We then relate the asymmetric fidelity at the replication fork to the efficiency of DNA mismatch repair, ribonucleotide excision repair and topoisomerase 1 activity.


Asunto(s)
Replicación del ADN , Inestabilidad Genómica , Disparidad de Par Base , Reparación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo
11.
Mol Cell ; 33(3): 287-98, 2009 Feb 13.
Artículo en Inglés | MEDLINE | ID: mdl-19217403

RESUMEN

Mis16 and Mis18 are subunits of a protein complex required for incorporation of the histone H3 variant CenH3 (Cnp1/CENP-A) into centromeric chromatin in Schizosaccharomyces pombe and mammals. How the Mis16-Mis18 complex performs this function is unknown. Here, we report that the Mis16-Mis18 complex is required for centromere localization of Scm3(Sp), a Cnp1-binding protein related to Saccharomyces cerevisiae Scm3. Scm3(Sp) is required for centromeric localization of Cnp1, while Scm3(Sp) localizes at centromeres independently of Cnp1. Like the Mis16-Mis18 complex but unlike Cnp1, Scm3(Sp) dissociates from centromeres during mitosis. Inactivation of Scm3(Sp) or Mis18 increases centromere localization of histones H3 and H2A/H2B, which are largely absent from centromeres in wild-type cells. Whereas S. cerevisiae Scm3 is proposed to replace histone H2A/H2B in centromeric nucleosomes, the dynamic behavior of S. pombe Scm3 suggests that it acts as a Cnp1 assembly/maintenance factor that directly mediates the stable deposition of Cnp1 into centromeric chromatin.


Asunto(s)
Proteínas Portadoras/metabolismo , Centrómero/metabolismo , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas Portadoras/análisis , Proteínas Portadoras/genética , Proteínas Cromosómicas no Histona/análisis , Histonas/metabolismo , Mitosis , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/análisis , Proteínas de Schizosaccharomyces pombe/genética
12.
PLoS Genet ; 8(10): e1003016, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23071460

RESUMEN

The two DNA strands of the nuclear genome are replicated asymmetrically using three DNA polymerases, α, δ, and ε. Current evidence suggests that DNA polymerase ε (Pol ε) is the primary leading strand replicase, whereas Pols α and δ primarily perform lagging strand replication. The fact that these polymerases differ in fidelity and error specificity is interesting in light of the fact that the stability of the nuclear genome depends in part on the ability of mismatch repair (MMR) to correct different mismatches generated in different contexts during replication. Here we provide the first comparison, to our knowledge, of the efficiency of MMR of leading and lagging strand replication errors. We first use the strand-biased ribonucleotide incorporation propensity of a Pol ε mutator variant to confirm that Pol ε is the primary leading strand replicase in Saccharomyces cerevisiae. We then use polymerase-specific error signatures to show that MMR efficiency in vivo strongly depends on the polymerase, the mismatch composition, and the location of the mismatch. An extreme case of variation by location is a T-T mismatch that is refractory to MMR. This mismatch is flanked by an AT-rich triplet repeat sequence that, when interrupted, restores MMR to > 95% efficiency. Thus this natural DNA sequence suppresses MMR, placing a nearby base pair at high risk of mutation due to leading strand replication infidelity. We find that, overall, MMR most efficiently corrects the most potentially deleterious errors (indels) and then the most common substitution mismatches. In combination with earlier studies, the results suggest that significant differences exist in the generation and repair of Pol α, δ, and ε replication errors, but in a generally complementary manner that results in high-fidelity replication of both DNA strands of the yeast nuclear genome.


Asunto(s)
Reparación de la Incompatibilidad de ADN , Replicación del ADN , Secuencia de Bases , ADN Polimerasa II/metabolismo , Datos de Secuencia Molecular , Mutagénesis , Tasa de Mutación , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
13.
EMBO J ; 29(6): 1136-48, 2010 Mar 17.
Artículo en Inglés | MEDLINE | ID: mdl-20094029

RESUMEN

ATM(Tel1) and ATR(Rad3) checkpoint kinases phosphorylate the C-terminus of histone H2AX (H2A in yeasts) in chromatin flanking DNA damage, establishing a recruitment platform for checkpoint and repair proteins. Phospho-H2A/X (gammaH2A/X)-binding proteins at double-strand breaks (DSBs) have been characterized, but those required for replication stress responses are unknown. Here, we present genetic, biochemical, small angle X-ray scattering (SAXS), and X-ray structural studies of the Schizosaccharomyces pombe Brc1, a 6-BRCT-domain protein that is structurally related to Saccharomyces cerevisiae Rtt107 and mammalian PTIP. Brc1 binds gammaH2A to form spontaneous and DNA damage-induced nuclear foci. Spontaneous Brc1 foci colocalize with ribosomal DNA repeats, a region prone to fork pausing and genomic instability, whereas DNA damage-induced Brc1 foci colocalize with DSB response factors. gammaH2A binding is critical for Brc1 function. The 1.45 A resolution crystal structure of Brc1-gammaH2A complex shows how variable BRCT insertion loops sculpt tandem-BRCT phosphoprotein-binding pockets to facilitate unique phosphoprotein-interaction specificities, and unveils an acidic DNA-mimicking Brc1 surface. From these results, Brc1 docking to gammaH2A emerges as a critical chromatin-specific response to replication-associated DNA damage.


Asunto(s)
Histonas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Cristalografía por Rayos X , Daño del ADN , Histonas/genética , Modelos Moleculares , Conformación Proteica , Fase S , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética
14.
Nat Commun ; 15(1): 8730, 2024 Oct 09.
Artículo en Inglés | MEDLINE | ID: mdl-39379399

RESUMEN

Finalization of eukaryotic nuclear DNA replication relies on DNA ligase 1 (LIG1) to seal DNA nicks generated during Okazaki Fragment Maturation (OFM). Using a mutational reporter in Saccharomyces cerevisiae, we previously showed that mutation of the high-fidelity magnesium binding site of LIG1Cdc9 strongly increases the rate of single-base insertions. Here we show that this rate is increased across the nuclear genome, that it is synergistically increased by concomitant loss of DNA mismatch repair (MMR), and that the additions occur in highly specific sequence contexts. These discoveries are all consistent with incorporation of an extra base into the nascent lagging DNA strand that can be corrected by MMR following mutagenic ligation by the Cdc9-EEAA variant. There is a strong preference for insertion of either dGTP or dTTP into 3-5 base pair mononucleotide sequences with stringent flanking nucleotide requirements. The results reveal unique LIG1Cdc9-dependent mutational motifs where high fidelity DNA ligation of a subset of OFs is critical for preventing mutagenesis across the genome.


Asunto(s)
ADN Ligasa (ATP) , Reparación de la Incompatibilidad de ADN , Replicación del ADN , ADN de Hongos , Genoma Fúngico , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Reparación de la Incompatibilidad de ADN/genética , ADN Ligasa (ATP)/genética , ADN Ligasa (ATP)/metabolismo , ADN de Hongos/genética , ADN de Hongos/metabolismo , Replicación del ADN/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , ADN/genética , ADN/metabolismo , Mutagénesis Insercional , Mutación , ADN Ligasas/metabolismo , ADN Ligasas/genética
15.
PLoS Genet ; 6(7): e1001032, 2010 Jul 22.
Artículo en Inglés | MEDLINE | ID: mdl-20661445

RESUMEN

Schizosaccharomyces pombe Rad3 checkpoint kinase and its human ortholog ATR are essential for maintaining genome integrity in cells treated with genotoxins that damage DNA or arrest replication forks. Rad3 and ATR also function during unperturbed growth, although the events triggering their activation and their critical functions are largely unknown. Here, we use ChIP-on-chip analysis to map genomic loci decorated by phosphorylated histone H2A (gammaH2A), a Rad3 substrate that establishes a chromatin-based recruitment platform for Crb2 and Brc1 DNA repair/checkpoint proteins. Unexpectedly, gammaH2A marks a diverse array of genomic features during S-phase, including natural replication fork barriers and a fork breakage site, retrotransposons, heterochromatin in the centromeres and telomeres, and ribosomal RNA (rDNA) repeats. gammaH2A formation at the centromeres and telomeres is associated with heterochromatin establishment by Clr4 histone methyltransferase. We show that gammaH2A domains recruit Brc1, a factor involved in repair of damaged replication forks. Brc1 C-terminal BRCT domain binding to gammaH2A is crucial in the absence of Rqh1(Sgs1), a RecQ DNA helicase required for rDNA maintenance whose human homologs are mutated in patients with Werner, Bloom, and Rothmund-Thomson syndromes that are characterized by cancer-predisposition or accelerated aging. We conclude that Rad3 phosphorylates histone H2A to mobilize Brc1 to critical genomic domains during S-phase, and this pathway functions in parallel with Rqh1 DNA helicase in maintaining genome integrity.


Asunto(s)
Proteínas de Ciclo Celular/fisiología , Cromosomas Fúngicos/metabolismo , Inestabilidad Genómica , Histonas/metabolismo , Proteínas Quinasas/fisiología , Fase S , Proteínas de Schizosaccharomyces pombe/fisiología , Schizosaccharomyces/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinasa de Punto de Control 2 , ADN Helicasas , Fosforilación , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo
16.
J Biol Chem ; 286(9): 7082-92, 2011 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-21190944

RESUMEN

Asf1 is a conserved histone H3/H4 chaperone. We find that Asf1 in budding yeast promotes an essential cellular response to replication stress caused by the ribonucleotide reductase inhibitor hydroxyurea. That is, Asf1 stimulates derepression of DNA damage response (DDR) genes during the S phase. Derepression of DDR genes strongly correlates with Asf1 binding to their promoters. Having identified the C terminus and histone-binding domains of Asf1 as molecular determinants of its constitutive and inducible association with chromatin, we tested whether Asf1 binding to DDR genes is mechanistically important for their derepression. Our results provide little support for this hypothesis. Rather, the contribution of Asf1 to DDR gene derepression depends on its ability to stimulate H3K56 acetylation by lysine acetyltransferase Rtt109. The precise regulation of H3K56 acetylation in the promoters of DDR genes is unexpected: DDR gene promoters are occupied by H3K56-acetylated nucleosomes under repressing conditions, and the steady state level of H3K56 promoter acetylation does not change upon derepression. We propose that replication-coupled deposition of Lys(56)-acetylated H3 poises the DDR genes in newly synthesized daughter duplexes for derepression during the S phase. In this model, the presence of a histone mark that destabilizes nucleosomes is compatible with suppression of transcription because in the uninduced state, DDR gene promoters are constitutively occupied by a potent repressor-corepressor complex.


Asunto(s)
Proteínas de Ciclo Celular/genética , Daño del ADN/fisiología , Chaperonas Moleculares/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Estrés Fisiológico/fisiología , Fosfatasa Ácida/genética , Fosfatasa Ácida/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/fisiología , Regulación Fúngica de la Expresión Génica , Biblioteca de Genes , Histona Acetiltransferasas/metabolismo , Histonas/metabolismo , Hidroxiurea/farmacología , Chaperonas Moleculares/metabolismo , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/fisiología , Estrés Fisiológico/efectos de los fármacos
17.
Cell Rep ; 41(1): 111448, 2022 10 04.
Artículo en Inglés | MEDLINE | ID: mdl-36198268

RESUMEN

Topoisomerase 1 (Top1) incises DNA containing ribonucleotides to generate complex DNA lesions that are resolved by APE2 (Apn2 in yeast). How Apn2 engages and processes this DNA damage is unclear. Here, we report X-ray crystal structures and biochemical analysis of Apn2-DNA complexes to demonstrate how Apn2 frays and cleaves 3' DNA termini via a wedging mechanism that facilitates 1-6 nucleotide endonucleolytic cleavages. APN2 deletion and DNA-wedge mutant Saccharomyces cerevisiae strains display mutator phenotypes, cell growth defects, and sensitivity to genotoxic stress in a ribonucleotide excision repair (RER)-defective background harboring a high density of Top1-incised ribonucleotides. Our data implicate a wedge-and-cut mechanism underpinning the broad-specificity Apn2 nuclease activity that mitigates mutagenic and genome instability phenotypes caused by Top1 incision at genomic ribonucleotides incorporated by DNA polymerase epsilon.


Asunto(s)
Proteínas de Saccharomyces cerevisiae , ADN , Daño del ADN , ADN Polimerasa II/genética , Reparación del ADN , ADN-Topoisomerasas de Tipo I/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Ribonucleótidos/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
18.
Nat Commun ; 12(1): 482, 2021 01 20.
Artículo en Inglés | MEDLINE | ID: mdl-33473124

RESUMEN

DNA ligase 1 (LIG1, Cdc9 in yeast) finalizes eukaryotic nuclear DNA replication by sealing Okazaki fragments using DNA end-joining reactions that strongly discriminate against incorrectly paired DNA substrates. Whether intrinsic ligation fidelity contributes to the accuracy of replication of the nuclear genome is unknown. Here, we show that an engineered low-fidelity LIG1Cdc9 variant confers a novel mutator phenotype in yeast typified by the accumulation of single base insertion mutations in homonucleotide runs. The rate at which these additions are generated increases upon concomitant inactivation of DNA mismatch repair, or by inactivation of the Fen1Rad27 Okazaki fragment maturation (OFM) nuclease. Biochemical and structural data establish that LIG1Cdc9 normally avoids erroneous ligation of DNA polymerase slippage products, and this protection is compromised by mutation of a LIG1Cdc9 high-fidelity metal binding site. Collectively, our data indicate that high-fidelity DNA ligation is required to prevent insertion mutations, and that this may be particularly critical following strand displacement synthesis during the completion of OFM.


Asunto(s)
Replicación del ADN/fisiología , ADN de Hongos/metabolismo , ADN/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetiltransferasas/metabolismo , ADN Ligasa (ATP)/metabolismo , ADN Ligasas , Reparación de la Incompatibilidad de ADN/genética , Replicación del ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Endonucleasas de ADN Solapado/metabolismo , Proteínas de la Membrana/metabolismo , Mutagénesis , Mutación , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
19.
Nat Struct Mol Biol ; 28(12): 1020-1028, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34887558

RESUMEN

Accurate DNA replication of an undamaged template depends on polymerase selectivity for matched nucleotides, exonucleolytic proofreading of mismatches, and removal of remaining mismatches via DNA mismatch repair (MMR). DNA polymerases (Pols) δ and ε have 3'-5' exonucleases into which mismatches are partitioned for excision in cis (intrinsic proofreading). Here we provide strong evidence that Pol δ can extrinsically proofread mismatches made by itself and those made by Pol ε, independently of both Pol δ's polymerization activity and MMR. Extrinsic proofreading across the genome is remarkably efficient. We report, with unprecedented accuracy, in vivo contributions of nucleotide selectivity, proofreading, and MMR to the fidelity of DNA replication in Saccharomyces cerevisiae. We show that extrinsic proofreading by Pol δ improves and balances the fidelity of the two DNA strands. Together, we depict a comprehensive picture of how nucleotide selectivity, proofreading, and MMR cooperate to achieve high and symmetrical fidelity on the two strands.


Asunto(s)
Reparación de la Incompatibilidad de ADN/genética , ADN Polimerasa III/metabolismo , ADN de Hongos/biosíntesis , Saccharomyces cerevisiae/genética , ADN Polimerasa II/metabolismo , Replicación del ADN/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
20.
DNA Repair (Amst) ; 84: 102641, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31311768

RESUMEN

Ribonucleotides are the most common non-canonical nucleotides incorporated into DNA during replication, and their processing leads to mutations and genome instability. Yeast mutation reporter systems demonstrate that 2-5 base pair deletions (Δ2-5bp) in repetitive DNA are a signature of unrepaired ribonucleotides, and that these events are initiated by topoisomerase 1 (Top1) cleavage. However, a detailed understanding of the frequency and locations of ribonucleotide-dependent mutational events across the genome has been lacking. Here we present the results of genome-wide mutational analysis of yeast strains deficient in Ribonucleotide Excision Repair (RER). We identified mutations that accumulated over thousands of generations in strains expressing either wild-type or variant replicase alleles (M644G Pol ε, L612M Pol δ, L868M Pol α) that confer increased ribonucleotide incorporation into DNA. Using a custom-designed mutation-calling pipeline called muver (for mutationes verificatae), we observe a number of surprising mutagenic features. This includes a 24-fold preferential elevation of AG and AC relative to AT dinucleotide deletions in the absence of RER, suggesting specificity for Top1-initiated deletion mutagenesis. Moreover, deletion rates in di- and trinucleotide repeat tracts increase exponentially with tract length. Consistent with biochemical and reporter gene mutational analysis, these deletions are no longer observed upon deletion of TOP1. Taken together, results from these analyses demonstrate the global impact of genomic ribonucleotide processing by Top1 on genome integrity.


Asunto(s)
Reparación del ADN , ADN-Topoisomerasas de Tipo I/metabolismo , Tasa de Mutación , Ribonucleótidos/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , ADN-Topoisomerasas de Tipo I/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Repeticiones de Dinucleótido , Eliminación de Gen , Inestabilidad Genómica , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Repeticiones de Trinucleótidos
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