RESUMO
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.
Assuntos
Replicação do DNA , Proteínas de Ligação a DNA , Proteína Homóloga a MRE11/genética , Proteína Homóloga a MRE11/metabolismo , Proteínas de Ligação a DNA/genética , Recombinação Homóloga/genética , DNARESUMO
Lynch syndrome (LS) is the main hereditary colorectal cancer syndrome. There have been few reports regarding the clinical and molecular characteristics of LS patients in Latin America; this is particularly true in the Mexican population, where no information is available. The present study aims to describe the clinical and molecular spectrum of variants in a cohort of patients diagnosed with LS in Mexico. We present a retrospective analysis of 412 patients with suspected LS, whose main site of cancer diagnosis was the colon (58.25%), followed by the endometrium (18.93%). Next-generation sequencing analysis, with an extensive multigene panel, showed that 27.1% (112/414) had a variant in one of the genes of the mismatch repair pathway (MMR); 30.4% (126/414) had a variant in non-MMR genes such as CHEK2, APC, MUTYH, BRCA1, and BRCA2; and 42.5% (176/414) had no genetic variants. Most of the variants were found in MLH1. Pathogenic variants (PVs) in MMR genes were identified in 65.7% (96/146) of the total PVs, and 34.24% (45/146) were in non-MMR genes. Molecular and clinical characterization of patients with LS in specific populations allowed personalized follow-up, with the option for targeted treatment with immune checkpoint inhibitors and the development of public health policies. Moreover, such characterization allows for family cascade testing and consequent prevention strategies.
Assuntos
Neoplasias Colorretais Hereditárias sem Polipose , Neoplasias Colorretais Hereditárias sem Polipose/diagnóstico , Neoplasias Colorretais Hereditárias sem Polipose/epidemiologia , Neoplasias Colorretais Hereditárias sem Polipose/genética , Reparo de Erro de Pareamento de DNA/genética , Proteínas de Ligação a DNA/genética , Feminino , Mutação em Linhagem Germinativa , Humanos , Inibidores de Checkpoint Imunológico , México/epidemiologia , Proteína 2 Homóloga a MutS/genética , Estudos RetrospectivosRESUMO
Although human nucleoporin Tpr is frequently deregulated in cancer, its roles are poorly understood. Here we show that Tpr depletion generates transcription-dependent replication stress, DNA breaks, and genomic instability. DNA fiber assays and electron microscopy visualization of replication intermediates show that Tpr deficient cells exhibit slow and asymmetric replication forks under replication stress. Tpr deficiency evokes enhanced levels of DNA-RNA hybrids. Additionally, complementary proteomic strategies identify a network of Tpr-interacting proteins mediating RNA processing, such as MATR3 and SUGP2, and functional experiments confirm that their depletion trigger cellular phenotypes shared with Tpr deficiency. Mechanistic studies reveal the interplay of Tpr with GANP, a component of the TREX-2 complex. The Tpr-GANP interaction is supported by their shared protein level alterations in a cohort of ovarian carcinomas. Our results reveal links between nucleoporins, DNA transcription and replication, and the existence of a network physically connecting replication forks with transcription, splicing, and mRNA export machinery.
Assuntos
Replicação do DNA , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismo , Sobrevivência Celular , Dano ao DNA , Instabilidade Genômica , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Mapas de Interação de Proteínas , Proteínas Proto-Oncogênicas/genética , Transporte de RNARESUMO
The deletion of exons 9 to 12 of BRCA1 (9-12 del BRCA1) is considered a founder mutation in the Mexican population. We evaluate the usefulness of the target detection of 9-12 del BRCA1 as the first molecular diagnostic strategy in patients with Hereditary Breast and Ovarian Cancer (HBOC). We performed the genetic assessment of 637 patients with suspected HBOC. The region corresponding to the breakpoints for the 9-12 del BRCA1 was amplified by polymerase chain reaction (PCR). An analysis of the clinical data of the carriers and non-carriers was done, searching for characteristics that correlated with the deletion. The 9-12 del BRCA1 was detected in 5% of patients with suspected HBOC (30/637). In patients diagnosed with ovarian cancer, 13 of 30 were 9-12 del BRCA1 carriers, which represents 43%. We found a significant association between the 9-12 del BRCA1 carriers with triple negative breast cancer and high-grade papillary serous ovarian cancer. We concluded that the detection of the 9-12 del BRCA1 is useful as a first molecular diagnostic strategy in the Mexican population. In particular, it shortens the gap in genetic assessment in patients with triple negative breast cancer and ovarian cancer.
