During S phase, the replisome has to overcome many physical obstacles that can cause replication fork stalling and compromise genome integrity. Transcription is an important source of replicative stress and consequently, maintenance of genome integrity requires the protection of chromosomes from the deleterious effects arising from the interaction between nascent RNAs and template DNA, leading to stable DNA-RNA hybrids (R-loop) formation. We previously reported the essential role of Omcg1 (Ovum Mutant Candidate Gene) for cell cycle progression during early embryonic development. Here, we show that OMCG1 is a target of the cell cycle checkpoint kinases ATR/ATM and is essential for S phase progression in mouse embryonic fibroblasts. Using a conditional gene inactivation strategy, we demonstrate that OMCG1 depletion impairs cell viability as a consequence of DSB formation, checkpoint activation and replication fork collapse. We also show that no chromosome breaks were generated in non-cycling Omcg1-deficient cells. Furthermore, increased RNaseH expression significantly alleviated genomic instability in deficient fibroblasts suggesting that cotranscriptional R-loops formation contributes to the genesis of replication-dependent DSBs in these cells. Together with recent reports describing its participation to complexes involved in cotanscriptional processes, our results suggest that OMCG1 plays a role in the tight coupling between mRNA processing pathways and maintenance of genome integrity during cell cycle progression.
"How these processes are linked and regulated in vivo remains largely unknown. In this study, we addressed in vivo the function of Omcg1, which has been shown to participate in co-transcriptional processes, including splicing and transcription-coupled repair (Bessonov et al., 2008; Kuraoka et al., 2008; Houlard et al., 2011). We showed that Omcg1 is most critically required in intestinal progenitors. "
[Show abstract][Hide abstract] ABSTRACT: Recent studies have shown that factors involved in transcription-coupled mRNA processing are important for the maintenance of genome integrity. How these processes are linked and regulated in vivo remains largely unknown. In this study, we addressed in the mouse model the function of Omcg1, which has been shown to participate in co-transcriptional processes, including splicing and transcription-coupled repair. Using inducible mouse models, we found that Omcg1 is most critically required in intestinal progenitors. In absence of OMCG1, proliferating intestinal epithelial cells underwent abnormal mitosis followed by apoptotic cell death. As a consequence, the crypt proliferative compartment of the small intestine was quickly and totally abrogated leading to the rapid death of the mice. Lack of OMCG1 in embryonic stem cells led to a similar cellular phenotype, with multiple mitotic defects and rapid cell death. We showed that mutant intestinal progenitors and embryonic stem cells exhibited a reduced cell cycle arrest following irradiation, suggesting that mitotic defects may be consecutive to M phase entry with unrepaired DNA damages. These findings unravel a crucial role for pre-mRNA processing in the homeostasis of the small intestine and point to a major role of OMCG1 in the maintenance of genome integrity.
Biology Open 07/2012; 1(7):648-57. DOI:10.1242/bio.20121248 · 2.42 Impact Factor
"Since co-transcriptional R-loops impair replication fork progression, an important question is whether these structures also activate the DNA replication checkpoint. This issue has recently been addressed in yeast and murine cells [99, 100]. In yeast hpr1 mutants, the Mec1-Rad53 pathway is constitutively activated, supporting the view that R-loops-mediated fork stalling activates the S-phase checkpoint. "
[Show abstract][Hide abstract] ABSTRACT: Replication and transcription are key aspects of DNA metabolism that take place on the same template and potentially interfere with each other. Conflicts between these two activities include head-on or co-directional collisions between DNA and RNA polymerases, which can lead to the formation of DNA breaks and chromosome rearrangements. To avoid these deleterious consequences and prevent genomic instability, cells have evolved multiple mechanisms preventing replication forks from colliding with the transcription machinery. Yet, recent reports indicate that interference between replication and transcription is not limited to physical interactions between polymerases and that other cotranscriptional processes can interfere with DNA replication. These include DNA-RNA hybrids that assemble behind elongating RNA polymerases, impede fork progression and promote homologous recombination. Here, we discuss recent evidence indicating that R-loops represent a major source of genomic instability in all organisms, from bacteria to human, and are potentially implicated in cancer development.
Current Genomics 03/2012; 13(1):65-73. DOI:10.2174/138920212799034767 · 2.34 Impact Factor
"In agreement, loss of ATR or ATR downstream targets is linked with replication fork collapse and CFS break formation (Casper et al., 2002; Cha and Kleckner, 2002; Durkin et al., 2006; Houlard et al., 2011). Thus, the known function of ATR to restart replication forks may be involved in preventing R-loop stabilization at collision sites. "
[Show abstract][Hide abstract] ABSTRACT: We show that the time required to transcribe human genes larger than 800 kb spans more than one complete cell cycle, while their transcription speed equals that of smaller genes. Independently of their expression status, we find the long genes to replicate late. Regions of concomitant transcription and replication in late S phase exhibit DNA break hot spots known as common fragile sites (CFSs). This CFS instability depends on the expression of the underlying long genes. We show that RNA:DNA hybrids (R-loops) form at sites of transcription/replication collisions and that RNase H1 functions to suppress CFS instability. In summary, our results show that, on the longest human genes, collisions of the transcription machinery with a replication fork are inevitable, creating R-loops and consequent CFS formation. Functional replication machinery needs to be involved in the resolution of conflicts between transcription and replication machineries to ensure genomic stability.
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