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1.
Nucleic Acids Res ; 37(10): 3177-88, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19305001

RESUMO

Repair of single-stranded DNA breaks before DNA replication is critical in maintaining genomic stability; however, how cells deal with these lesions during S phase is not clear. Using combined approaches of proteomics and in vitro and in vivo protein-protein interaction, we identified the p58 subunit of DNA Pol alpha-primase as a new binding partner of XRCC1, a key protein of the single strand break repair (SSBR) complex. In vitro experiments reveal that the binding of poly(ADP-ribose) to p58 inhibits primase activity by competition with its DNA binding property. Overexpression of the XRCC1-BRCT1 domain in HeLa cells induces poly(ADP-ribose) synthesis, PARP-1 and XRCC1-BRCT1 poly(ADP-ribosyl)ation and a strong S phase delay in the presence of DNA damage. Addition of recombinant XRCC1-BRCT1 to Xenopus egg extracts slows down DNA synthesis and inhibits the binding of PCNA, but not MCM2 to alkylated chromatin, thus indicating interference with the assembly of functional replication forks. Altogether these results suggest a critical role for XRCC1 in connecting the SSBR machinery with the replication fork to halt DNA synthesis in response to DNA damage.


Assuntos
DNA Primase/metabolismo , Reparo do DNA , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Fase S/genética , Animais , Cromatina/metabolismo , DNA/biossíntese , Dano ao DNA , DNA Polimerase I/metabolismo , DNA Primase/química , Proteínas de Ligação a DNA/química , Células HeLa , Humanos , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteína 1 Complementadora Cruzada de Reparo de Raio-X , Xenopus laevis
2.
Adv Exp Med Biol ; 604: 3-16, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17695718

RESUMO

DNA replication is a fundamental process within the cell cycle. The exact duplication of the genetic information ensures genome stability. Extensive research has identified the principal players required for the sequential processes: origin-licensing (a controlled order of events giving a chromosome site the potential to be initiated within the S phase of the same cell cycle); initiation (by removing the license a previous licensed site is transformed into a site where the DNA helix starts to melt); and DNA replication (copying the parental DNA by leading and lagging strand DNA-synthesis). The present report compares the advantages and limitations of studying DNA replication in the model systems Xenopus laevis (X. laevis) and in Simian Virus 40 (SV40).


Assuntos
Replicação do DNA , Vírus 40 dos Símios/metabolismo , Xenopus laevis/metabolismo , Animais , Sítios de Ligação , Ciclo Celular , DNA/química , Humanos , Complexo de Reconhecimento de Origem , Origem de Replicação , Fase S , Especificidade da Espécie
3.
J Cell Biol ; 173(5): 673-83, 2006 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-16754955

RESUMO

In late mitosis and early G1, replication origins are licensed for subsequent use by loading complexes of the minichromosome maintenance proteins 2-7 (Mcm2-7). The number of Mcm2-7 complexes loaded onto DNA greatly exceeds the number of replication origins used during S phase, but the function of the excess Mcm2-7 is unknown. Using Xenopus laevis egg extracts, we show that these excess Mcm2-7 complexes license additional dormant origins that do not fire during unperturbed S phases because of suppression by a caffeine-sensitive checkpoint pathway. Use of these additional origins can allow complete genome replication in the presence of replication inhibitors. These results suggest that metazoan replication origins are actually comprised of several candidate origins, most of which normally remain dormant unless cells experience replicative stress. Consistent with this model, using Caenorhabditis elegans, we show that partial RNAi-based knockdown of MCMs that has no observable effect under normal conditions causes lethality upon treatment with low, otherwise nontoxic, levels of the replication inhibitor hydroxyurea.


Assuntos
Replicação do DNA/fisiologia , Estresse Oxidativo/fisiologia , Origem de Replicação , Proteínas de Xenopus/metabolismo , Adenosina Trifosfatases/efeitos dos fármacos , Adenosina Trifosfatases/metabolismo , Animais , Afidicolina/farmacologia , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/crescimento & desenvolvimento , Cafeína/farmacologia , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Replicação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Hidroxiureia/farmacologia , Componente 2 do Complexo de Manutenção de Minicromossomo , Componente 3 do Complexo de Manutenção de Minicromossomo , Componente 4 do Complexo de Manutenção de Minicromossomo , Componente 7 do Complexo de Manutenção de Minicromossomo , Proteínas Nucleares/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Fatores de Tempo , Proteínas de Xenopus/efeitos dos fármacos , Xenopus laevis
4.
J Cell Sci ; 117(Pt 25): 6019-30, 2004 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-15536124

RESUMO

In most eukaryotes, replication origins fire asynchronously throughout S-phase according to a precise timing programme. When replication fork progression is inhibited, an intra-S-phase checkpoint is activated that blocks further origin firing and stabilizes existing replication forks to prevent them undergoing irreversible collapse. We show that chromatin incubated in Xenopus egg extracts displays a replication-timing programme in which firing of new replication origins during S phase depends on the continued activity of S-phase-inducing cyclin-dependent kinases. We also show that low concentrations of the DNA-polymerase inhibitor aphidicolin, which only slightly slows replication-fork progression, strongly suppress further initiation events. This intra-S-phase checkpoint can be overcome by caffeine, an inhibitor of the ATM/ATR checkpoint kinases, or by neutralizing antibodies to ATR. However, depletion or inhibition of Chk1 did not abolish the checkpoint. We could detect no significant effect on fork stability when this intra-S-phase checkpoint was inhibited. Interestingly, although caffeine could prevent the checkpoint from being activated, it could not rescue replication if added after the timing programme would normally have been executed. This suggests that special mechanisms might be necessary to reverse the effects of the intra-S-phase checkpoint once it has acted on particular origins.


Assuntos
Proteínas de Ciclo Celular/fisiologia , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas de Xenopus/fisiologia , Animais , Afidicolina/metabolismo , Afidicolina/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia , Cafeína/metabolismo , Cafeína/farmacologia , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Quinase 1 do Ponto de Checagem , Cromatina/metabolismo , Ciclina A/farmacologia , Replicação do DNA , Eletroforese em Gel de Ágar , Inibidores Enzimáticos/farmacologia , Cinética , Masculino , Inibidores da Síntese de Ácido Nucleico , Antígeno Nuclear de Célula em Proliferação/metabolismo , Purinas/farmacologia , Roscovitina , Fase S , Espermatozoides/metabolismo , Fatores de Tempo , Xenopus , Proteínas de Xenopus/metabolismo
5.
J Cell Biol ; 165(2): 181-90, 2004 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-15096526

RESUMO

Before S phase, cells license replication origins for initiation by loading them with Mcm2-7 heterohexamers. This process is dependent on Cdc6, which is recruited to unlicensed origins. Using Xenopus egg extracts we show that although each origin can load many Mcm2-7 hexamers, the affinity of Cdc6 for each origins drops once it has been licensed by loading the first hexamers. This encourages the distribution of at least one Mcm2-7 hexamer to each origin, and thereby helps to ensure that all origins are licensed. Although Cdc6 is not essential for DNA replication once licensing is complete, Cdc6 regains a high affinity for origins once replication forks are initiated and Mcm2-7 has been displaced from the origin DNA. We show that the presence of Cdc6 during S phase is essential for the checkpoint kinase Chk1 to become activated in response to replication inhibition. These results show that Cdc6 plays multiple roles in ensuring precise chromosome duplication.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Replicação do DNA , Genoma , Subunidades Proteicas/metabolismo , Fase S/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Animais , Afidicolina/metabolismo , Quinase 1 do Ponto de Checagem , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Inibidores Enzimáticos/metabolismo , Feminino , Humanos , Masculino , Modelos Genéticos , Proteínas Nucleares/metabolismo , Oócitos/fisiologia , Complexo de Reconhecimento de Origem , Ligação Proteica , Proteínas Quinases/metabolismo , Estrutura Quaternária de Proteína , Origem de Replicação , Espermatozoides/metabolismo , Proteínas de Xenopus , Xenopus laevis
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