Rrm3 and Pif1 division of labor during replication through leading and lagging strand G-quadruplex.

Members of the conserved Pif1 family of 5'-3' DNA helicases can unwind G4s and mitigate their negative impact on genome stability. In Saccharomyces cerevisiae, two Pif1 family members, Pif1 and Rrm3, contribute to the suppression of genomic instability at diverse regions including telomeres, centromeres and tRNA genes. While Pif1 can resolve lagging strand G4s in vivo, little is known regarding Rrm3 function at G4s and its cooperation with Pif1 for G4 replication. Here, we monitored replication through G4 sequences in real time to show that Rrm3 is essential for efficient replisome progression through G4s located on the leading strand template, but not on the lagging strand. We found that Rrm3 importance for replication through G4s is dependent on its catalytic activity and its N-terminal unstructured region. Overall, we show that Rrm3 and Pif1 exhibit a division of labor that enables robust replication fork progression through leading and lagging strand G4s, respectively.

Impact of R-loops on oncogene-induced replication stress in cancer cells.

Replication stress is an alteration in the progression of replication forks caused by a variety of events of endogenous or exogenous origin. In precancerous lesions, this stress is exacerbated by the deregulation of oncogenic pathways, which notably disrupts the coordination between replication and transcription, and leads to genetic instability and cancer development. It is now well established that transcription can interfere with genome replication in different ways, such as head-on collisions between polymerases, accumulation of positive DNA supercoils or formation of R-loops. These structures form during transcription when nascent RNA reanneals with DNA behind the RNA polymerase, forming a stable DNA:RNA hybrid. In this review, we discuss how these different cotranscriptional processes disrupt the progression of replication forks and how they contribute to genetic instability in cancer cells.

Le stress réplicatif correspond à une altération de la progression des fourches de réplication causé par une variété d'événements d'origine endogène ou exogène. Dans les lésions précancéreuses, ce stress est aggravé par la dérégulation de voies oncogéniques, qui perturbe notamment la coordination entre la réplication et la transcription du génome et entraine une instabilité génétique contribuant au développement du cancer. Il est maintenant bien établi que la transcription peut interférer avec la réplication du génome de différentes façons, telles que des collisions frontales entre polymérases, l'accumulation de supertours positifs de l'ADN ou la formation de R-loops. Ces structures se forment au cours de la transcription lorsque l'ARN naissant se réassocie avec l'ADN derrière l'ARN polymérase, formant un hybride ADN :ARN stable. Dans cette revue, nous discutons comment ces différents processus cotranscriptionnels perturbent la progression des fourches de réplication et comment ils contribuent à l'instabilité génétique des cellules cancéreuses.

RNase H2 degrades toxic RNA:DNA hybrids behind stalled forks to promote replication restart.

R-loops represent a major source of replication stress, but the mechanism by which these structures impede fork progression remains unclear. To address this question, we monitored fork progression, arrest, and restart in Saccharomyces cerevisiae cells lacking RNase H1 and H2, two enzymes responsible for degrading RNA:DNA hybrids. We found that while RNase H-deficient cells could replicate their chromosomes normally under unchallenged growth conditions, their replication was impaired when exposed to hydroxyurea (HU) or methyl methanesulfonate (MMS). Treated cells exhibited increased levels of RNA:DNA hybrids at stalled forks and were unable to generate RPA-coated single-stranded (ssDNA), an important postreplicative intermediate in resuming replication. Similar impairments in nascent DNA resection and ssDNA formation at HU-arrested forks were observed in human cells lacking RNase H2. However, fork resection was fully restored by addition of triptolide, an inhibitor of transcription that induces RNA polymerase degradation. Taken together, these data indicate that RNA:DNA hybrids not only act as barriers to replication forks, but also interfere with postreplicative fork repair mechanisms if not promptly degraded by RNase H.