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.

Essential Roles of the Smc5/6 Complex in Replication through Natural Pausing Sites and Endogenous DNA Damage Tolerance.

The essential functions of the conserved Smc5/6 complex remain elusive. To uncover its roles in genome maintenance, we established Saccharomyces cerevisiae cell-cycle-regulated alleles that enable restriction of Smc5/6 components to S or G2/M. Unexpectedly, the essential functions of Smc5/6 segregated fully and selectively to G2/M. Genetic screens that became possible with generated alleles identified processes that crucially rely on Smc5/6 specifically in G2/M: metabolism of DNA recombination structures triggered by endogenous replication stress, and replication through natural pausing sites located in late-replicating regions. In the first process, Smc5/6 modulates remodeling of recombination intermediates, cooperating with dissolution activities. In the second, Smc5/6 prevents chromosome fragility and toxic recombination instigated by prolonged pausing and the fork protection complex, Tof1-Csm3. Our results thus dissect Smc5/6 essential roles and reveal that combined defects in DNA damage tolerance and pausing site-replication cause recombination-mediated DNA lesions, which we propose to drive developmental and cancer-prone disorders.

Closing the MCM cycle at replication termination sites.

The initiation of eukaryotic DNA replication is a highly regulated process conserved from yeast to human. The past decade has seen significant advances in understanding how the CMG (Cdc45­MCM­GINS) replicative helicase is loaded onto DNA. However, very little was known on how this complex is removed from chromatin at the end of S phase. Two papers in a recent issue of Science [1], [2] show that in yeast and in Xenopus, the CMG complex is unloaded at replication termination sites by an active mechanism involving the polyubiquitylation of Mcm7.

dNTP pools determine fork progression and origin usage under replication stress.

Intracellular deoxyribonucleoside triphosphate (dNTP) pools must be tightly regulated to preserve genome integrity. Indeed, alterations in dNTP pools are associated with increased mutagenesis, genomic instability and tumourigenesis. However, the mechanisms by which altered or imbalanced dNTP pools affect DNA synthesis remain poorly understood. Here, we show that changes in intracellular dNTP levels affect replication dynamics in budding yeast in different ways. Upregulation of the activity of ribonucleotide reductase (RNR) increases elongation, indicating that dNTP pools are limiting for normal DNA replication. In contrast, inhibition of RNR activity with hydroxyurea (HU) induces a sharp transition to a slow-replication mode within minutes after S-phase entry. Upregulation of RNR activity delays this transition and modulates both fork speed and origin usage under replication stress. Interestingly, we also observed that chromosomal instability (CIN) mutants have increased dNTP pools and show enhanced DNA synthesis in the presence of HU. Since upregulation of RNR promotes fork progression in the presence of DNA lesions, we propose that CIN mutants adapt to chronic replication stress by upregulating dNTP pools.

Establishment of sister chromatid cohesion at the S. cerevisiae replication fork.

Two identical sister copies of eukaryotic chromosomes are synthesized during S phase. To facilitate their recognition as pairs for segregation in mitosis, sister chromatids are held together from their synthesis onward by the chromosomal cohesin complex. Replication fork progression is thought to be coupled to establishment of sister chromatid cohesion, facilitating identification of replication products, but evidence for this has remained circumstantial. Here we show that three proteins required for sister chromatid cohesion, Eco1, Ctf4, and Ctf18, are found at, and Ctf4 travels along chromosomes with, replication forks. The ring-shaped cohesin complex is loaded onto chromosomes before S phase in an ATP hydrolysis-dependent reaction. Cohesion establishment during DNA replication follows without further cohesin recruitment and without need for cohesin to re-engage an ATP hydrolysis motif that is critical for its initial DNA binding. This provides evidence for cohesion establishment in the context of replication forks and imposes constraints on the mechanism involved.

The yeast CDK inhibitor Sic1 prevents genomic instability by promoting replication origin licensing in late G(1).

G(1) cell cycle regulators are often mutated in cancer, but how this causes genomic instability is unclear. Here we show that yeast lacking the CDK inhibitor Sic1 initiate DNA replication from fewer origins, have an extended S phase, and inefficiently separate sister chromatids during anaphase. This leads to double-strand breaks (DSBs) in a fraction of sic1 cells as evidenced by the accumulation of Ddc1 foci and a 575-fold increase in gross chromosomal rearrangements. Both S and M phase defects are rescued by delaying S-CDK activation, indicating that Sic1 promotes origin licensing in late G(1) by preventing the untimely activation of CDKs. We propose that precocious CDK activation causes genomic instability by altering the dynamics of S phase, which then hinders normal chromosome segregation.