RESUMO
R-loops have both positive and negative impacts on chromosome functions. To identify toxic R-loops in the human genome, here, we map RNA:DNA hybrids, replication stress markers and DNA double-strand breaks (DSBs) in cells depleted for Topoisomerase I (Top1), an enzyme that relaxes DNA supercoiling and prevents R-loop formation. RNA:DNA hybrids are found at both promoters (TSS) and terminators (TTS) of highly expressed genes. In contrast, the phosphorylation of RPA by ATR is only detected at TTS, which are preferentially replicated in a head-on orientation relative to the direction of transcription. In Top1-depleted cells, DSBs also accumulate at TTS, leading to persistent checkpoint activation, spreading of γ-H2AX on chromatin and global replication fork slowdown. These data indicate that fork pausing at the TTS of highly expressed genes containing R-loops prevents head-on conflicts between replication and transcription and maintains genome integrity in a Top1-dependent manner.
Assuntos
Replicação do DNA , DNA Topoisomerases Tipo I/metabolismo , Estruturas R-Loop/genética , Regiões Terminadoras Genéticas/genética , Transcrição Gênica , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Topoisomerases Tipo I/genética , Técnicas de Silenciamento de Genes , Instabilidade Genômica , Células HEK293 , Células HeLa , Humanos , Fosforilação , Regiões Promotoras Genéticas , RNA Interferente Pequeno/metabolismoRESUMO
Oncogene-induced replication stress (RS) promotes cancer development but also impedes tumor growth by activating anti-cancer barriers. To determine how cancer cells adapt to RS, we have monitored the expression of different components of the ATR-CHK1 pathway in primary tumor samples. We show that unlike upstream components of the pathway, the checkpoint mediators Claspin and Timeless are overexpressed in a coordinated manner. Remarkably, reducing the levels of Claspin and Timeless in HCT116 cells to pretumoral levels impeded fork progression without affecting checkpoint signaling. These data indicate that high level of Claspin and Timeless increase RS tolerance by protecting replication forks in cancer cells. Moreover, we report that primary fibroblasts adapt to oncogene-induced RS by spontaneously overexpressing Claspin and Timeless, independently of ATR signaling. Altogether, these data indicate that enhanced levels of Claspin and Timeless represent a gain of function that protects cancer cells from of oncogene-induced RS in a checkpoint-independent manner.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Adenocarcinoma de Pulmão/patologia , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular/biossíntese , Neoplasias Colorretais/patologia , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Estresse Fisiológico/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Adenocarcinoma de Pulmão/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Neoplasias da Mama/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/metabolismo , Neoplasias Colorretais/genética , Dano ao DNA/genética , Instabilidade Genômica/genética , Células HCT116 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Células MCF-7 , Estresse Fisiológico/genéticaRESUMO
SAMHD1 was previously characterized as a dNTPase that protects cells from viral infections. Mutations in SAMHD1 are implicated in cancer development and in a severe congenital inflammatory disease known as Aicardi-Goutières syndrome. The mechanism by which SAMHD1 protects against cancer and chronic inflammation is unknown. Here we show that SAMHD1 promotes degradation of nascent DNA at stalled replication forks in human cell lines by stimulating the exonuclease activity of MRE11. This function activates the ATR-CHK1 checkpoint and allows the forks to restart replication. In SAMHD1-depleted cells, single-stranded DNA fragments are released from stalled forks and accumulate in the cytosol, where they activate the cGAS-STING pathway to induce expression of pro-inflammatory type I interferons. SAMHD1 is thus an important player in the replication stress response, which prevents chronic inflammation by limiting the release of single-stranded DNA from stalled replication forks.
