RESUMO
It has been proposed that ATR kinase senses the completion of DNA replication to initiate the S/G2 transition. In contrast to this model, we show here that the TRESLIN-MTBP complex prevents a premature entry into G2 from early S-phase independently of ATR/CHK1 kinases. TRESLIN-MTBP acts transiently at pre-replication complexes (preRCs) to initiate origin firing and is released after the subsequent recruitment of CDC45. This dynamic behavior of TRESLIN-MTBP implements a monitoring system that checks the activation of replication forks and senses the rate of origin firing to prevent the entry into G2. This system detects the decline in the number of origins of replication that naturally occurs in very late S, which is the signature that cells use to determine the completion of DNA replication and permit the S/G2 transition. Our work introduces TRESLIN-MTBP as a key player in cell-cycle control independent of canonical checkpoints.
Assuntos
Proteínas de Ciclo Celular , Replicação do DNA , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase 1 do Ponto de Checagem/genética , Proteínas de Ligação a DNA/genéticaRESUMO
BRCA1 or BRCA2 germline mutations predispose to breast, ovarian and other cancers. High-throughput sequencing of tumour genomes revealed that oncogene amplification and BRCA1/2 mutations are mutually exclusive in cancer, however the molecular mechanism underlying this incompatibility remains unknown. Here, we report that activation of ß-catenin, an oncogene of the WNT signalling pathway, inhibits proliferation of BRCA1/2-deficient cells. RNA-seq analyses revealed ß-catenin-induced discrete transcriptome alterations in BRCA2-deficient cells, including suppression of CDKN1A gene encoding the CDK inhibitor p21. This accelerates G1/S transition, triggering illegitimate origin firing and DNA damage. In addition, ß-catenin activation accelerates replication fork progression in BRCA2-deficient cells, which is critically dependent on p21 downregulation. Importantly, we find that upregulated p21 expression is essential for the survival of BRCA2-deficient cells and tumours. Thus, our work demonstrates that ß-catenin toxicity in cancer cells with compromised BRCA1/2 function is driven by transcriptional alterations that cause aberrant replication and inflict DNA damage.
Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Oncogenes/genética , Transcrição Gênica/genética , beta Catenina/genética , Proteína BRCA1/deficiência , Proteína BRCA2/deficiência , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA , Feminino , Perfilação da Expressão Gênica/métodos , Células HeLa , Humanos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , RNA-Seq/métodos , beta Catenina/metabolismoRESUMO
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.
Assuntos
Replicação do DNA/genética , DNA de Cadeia Simples/genética , Ligação Proteica/genética , HumanosRESUMO
Maintenance of genome integrity requires the functional interplay between Fanconi anemia (FA) and homologous recombination (HR) repair pathways. Endogenous acetaldehyde, a product of cellular metabolism, is a potent source of DNA damage, particularly toxic to cells and mice lacking the FA protein FANCD2. Here, we investigate whether HR-compromised cells are sensitive to acetaldehyde, similarly to FANCD2-deficient cells. We demonstrate that inactivation of HR factors BRCA1, BRCA2, or RAD51 hypersensitizes cells to acetaldehyde treatment, in spite of the FA pathway being functional. Aldehyde dehydrogenases (ALDHs) play key roles in endogenous acetaldehyde detoxification, and their chemical inhibition leads to cellular acetaldehyde accumulation. We find that disulfiram (Antabuse), an ALDH2 inhibitor in widespread clinical use for the treatment of alcoholism, selectively eliminates BRCA1/2-deficient cells. Consistently, Aldh2 gene inactivation suppresses proliferation of HR-deficient mouse embryonic fibroblasts (MEFs) and human fibroblasts. Hypersensitivity of cells lacking BRCA2 to acetaldehyde stems from accumulation of toxic replication-associated DNA damage, leading to checkpoint activation, G2/M arrest, and cell death. Acetaldehyde-arrested replication forks require BRCA2 and FANCD2 for protection against MRE11-dependent degradation. Importantly, acetaldehyde specifically inhibits in vivo the growth of BRCA1/2-deficient tumors and ex vivo in patient-derived tumor xenograft cells (PDTCs), including those that are resistant to poly (ADP-ribose) polymerase (PARP) inhibitors. The work presented here therefore identifies acetaldehyde metabolism as a potential therapeutic target for the selective elimination of BRCA1/2-deficient cells and tumors.