RESUMEN
POLθ promotes repair of DNA double-strand breaks (DSBs) resulting from collapsed forks in homologous recombination (HR) defective tumors. Inactivation of POLθ results in synthetic lethality with the loss of HR genes BRCA1/2, which induces under-replicated DNA accumulation. However, it is unclear whether POLθ-dependent DNA replication prevents HR-deficiency-associated lethality. Here, we isolated Xenopus laevis POLθ and showed that it processes stalled Okazaki fragments, directly visualized by electron microscopy, thereby suppressing ssDNA gaps accumulating on lagging strands in the absence of RAD51 and preventing fork reversal. Inhibition of POLθ DNA polymerase activity leaves fork gaps unprotected, enabling their cleavage by the MRE11-NBS1-CtIP endonuclease, which produces broken forks with asymmetric single-ended DSBs, hampering BRCA2-defective cell survival. These results reveal a POLθ-dependent genome protection function preventing stalled forks rupture and highlight possible resistance mechanisms to POLθ inhibitors.
Asunto(s)
Replicación del ADN , Proteínas de Unión al ADN , Proteína Homóloga de MRE11/genética , Proteína Homóloga de MRE11/metabolismo , Proteínas de Unión al ADN/genética , Recombinación Homóloga/genética , ADNRESUMEN
Brca2 deficiency causes Mre11-dependent degradation of nascent DNA at stalled forks, leading to cell lethality. To understand the molecular mechanisms underlying this process, we isolated Xenopus laevis Brca2. We demonstrated that Brca2 protein prevents single-stranded DNA gap accumulation at replication fork junctions and behind them by promoting Rad51 binding to replicating DNA. Without Brca2, forks with persistent gaps are converted by Smarcal1 into reversed forks, triggering extensive Mre11-dependent nascent DNA degradation. Stable Rad51 nucleofilaments, but not RPA or Rad51T131P mutant proteins, directly prevent Mre11-dependent DNA degradation. Mre11 inhibition instead promotes reversed fork accumulation in the absence of Brca2. Rad51 directly interacts with the Pol α N-terminal domain, promoting Pol α and δ binding to stalled replication forks. This interaction likely promotes replication fork restart and gap avoidance. These results indicate that Brca2 and Rad51 prevent formation of abnormal DNA replication intermediates, whose processing by Smarcal1 and Mre11 predisposes to genome instability.
Asunto(s)
Proteína BRCA2/metabolismo , Replicación del ADN , ADN/biosíntesis , Recombinasa Rad51/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animales , Proteína BRCA2/genética , Sitios de Unión , ADN/genética , ADN Helicasas/genética , ADN Helicasas/metabolismo , ADN Polimerasa I/metabolismo , ADN Polimerasa III/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Femenino , Inestabilidad Genómica , Humanos , Proteína Homóloga de MRE11 , Masculino , Mutación , Unión Proteica , Recombinasa Rad51/genética , Origen de Réplica , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Tiempo , Proteínas de Xenopus/genética , Xenopus laevis/genéticaRESUMEN
It has been long assumed that normally leading strand synthesis must proceed coordinated with the lagging strand to prevent strand uncoupling and the pathological accumulation of single-stranded DNA (ssDNA) in the cell, a dogma recently challenged by in vitro studies in prokaryotes. Here, we report that human DNA polymerases can function independently at each strand in vivo and that the resulting strand uncoupling is supported physiologically by a cellular tolerance to ssDNA. Active forks rapidly accumulate ssDNA at the lagging strand when POLA1 is inhibited without triggering a stress response, despite ssDNA formation being considered a hallmark of replication stress. Acute POLA1 inhibition causes a lethal RPA exhaustion, but cells can duplicate their DNA with limited POLA1 activity and exacerbated strand uncoupling as long as RPA molecules suffice to protect the elevated ssDNA. Although robust, this uncoupled mode of DNA replication is also an in-built weakness that can be targeted for cancer treatment.
Asunto(s)
Replicación del ADN/genética , ADN de Cadena Simple/genética , Unión Proteica/genética , HumanosRESUMEN
Coordination between DNA replication and DNA repair ensures maintenance of genome integrity, which is lost in cancer cells. Emerging evidence has linked homologous recombination (HR) proteins RAD51, BRCA1 and BRCA2 to the stability of nascent DNA. This function appears to be distinct from double-strand break (DSB) repair and is in part due to the prevention of MRE11-mediated degradation of nascent DNA at stalled forks. The role of RAD51 in fork protection resembles the activity described for its prokaryotic orthologue RecA, which prevents nuclease-mediated degradation of DNA and promotes replication fork restart in cells challenged by DNA-damaging agents. Here, we examine the mechanistic aspects of HR-mediated fork protection, addressing the crosstalk between HR and replication proteins.
Asunto(s)
Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Replicación del ADN , Proteínas de Unión al ADN/antagonistas & inhibidores , Recombinación Homóloga , Modelos Biológicos , Recombinasa Rad51/metabolismo , Ácido Anhídrido Hidrolasas , Animales , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Inestabilidad Cromosómica , Roturas del ADN , Enzimas Reparadoras del ADN/química , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Humanos , Proteína Homóloga de MRE11 , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Multimerización de Proteína , Proteína de Replicación A/antagonistas & inhibidores , Proteína de Replicación A/química , Proteína de Replicación A/metabolismoRESUMEN
The correct duplication of genetic information is essential to maintain genome stability, which is lost in cancer cells. Replication fork integrity is ensured by a number of DNA metabolism proteins that assist replication of chromatin regions difficult to replicate due to their intrinsic DNA sequence composition, coordinate repair of DNA molecules resulting from aberrant replication events or protect replication forks in the presence of lesions impairing their progression. Some DNA metabolism genes involved in DNA repair are essential in higher eukaryotes even in unchallenged conditions, suggesting the existence of biological processes requiring these specialized functions in organisms with complex genomes. The impact on cell survival of null mutants of many DNA metabolism genes has precluded complete in depth analysis of their function. Cell free extracts represent a fundamental tool to overcome survival issues. The Xenopus laevis egg cell free extract is an ideal system to study replication-associated functions of essential genes. We are taking advantage of this system together with innovative imaging and proteomic based experimental approaches to characterize the molecular function of essential DNA metabolism proteins. Using this approach we have uncovered the role of some essential homologous recombination and fork protection proteins in chromosomal DNA replication and we have characterized some of the factors required for faithful replication of specific vertebrate genomic regions. This approach will be instrumental to study the molecular mechanisms underlying the function of a number of essential DNA metabolism proteins involved in the maintenance of genome stability in complex genomes.