In vivo reduction of RAD51-mediated homologous recombination triggers aging but impairs oncogenesis.

Homologous recombination (HR) is a prominent DNA repair pathway maintaining genome integrity. Mutations in many HR genes lead to cancer predisposition. Paradoxically, the implication of the pivotal HR factor RAD51 on cancer development remains puzzling. Particularly, no RAD51 mouse models are available to address the role of RAD51 in aging and carcinogenesis in vivo. We engineered a mouse model with an inducible dominant-negative form of RAD51 (SMRad51) that suppresses RAD51-mediated HR without stimulating alternative mutagenic repair pathways. We found that in vivo expression of SMRad51 led to replicative stress, systemic inflammation, progenitor exhaustion, premature aging and reduced lifespan, but did not trigger tumorigenesis. Expressing SMRAD51 in a breast cancer predisposition mouse model (PyMT) decreased the number and the size of tumors, revealing an anti-tumor activity of SMRAD51. We propose that these in vivo phenotypes result from chronic endogenous replication stress caused by HR decrease, which preferentially targets progenitors and tumor cells. Our work underlines the importance of RAD51 activity for progenitor cell homeostasis, preventing aging and more generally for the balance between cancer and aging.

Genome-wide Control of Heterochromatin Replication by the Telomere Capping Protein TRF2.

Hard-to-replicate regions of chromosomes (e.g., pericentromeres, centromeres, and telomeres) impede replication fork progression, eventually leading, in the event of replication stress, to chromosome fragility, aging, and cancer. Our knowledge of the mechanisms controlling the stability of these regions is essentially limited to telomeres, where fragility is counteracted by the shelterin proteins. Here we show that the shelterin subunit TRF2 ensures progression of the replication fork through pericentromeric heterochromatin, but not centromeric chromatin. In a process involving its N-terminal basic domain, TRF2 binds to pericentromeric Satellite III sequences during S phase, allowing the recruitment of the G-quadruplex-resolving helicase RTEL1 to facilitate fork progression. We also show that TRF2 is required for the stability of other heterochromatic regions localized throughout the genome, paving the way for future research on heterochromatic replication and its relationship with aging and cancer.

The impact of replication stress on replication dynamics and DNA damage in vertebrate cells.

The interplay between replication stress and the S phase checkpoint is a key determinant of genome maintenance, and has a major impact on human diseases, notably, tumour initiation and progression. Recent studies have yielded insights into sequence-dependent and sequence-independent sources of endogenous replication stress. These stresses result in nuclease-induced DNA damage, checkpoint activation and genome-wide replication fork slowing. Several hypotheses have been proposed to account for the mechanisms involved in this complex response. Recent results have shown that the slowing of the replication forks most commonly results from DNA precursor starvation. By concomitantly increasing the density of replication initiation, the cell elicits an efficient compensatory strategy to avoid mitotic anomalies and the inheritance of damage over cell generations.

Firing of Replication Origins Is Disturbed by a CDK4/6 Inhibitor in a pRb-Independent Manner.

Over the last decade, CDK4/6 inhibitors (palbociclib, ribociclib and abemaciclib) have emerged as promising anticancer drugs. Numerous studies have demonstrated that CDK4/6 inhibitors efficiently block the pRb-E2F pathway and induce cell cycle arrest in pRb-proficient cells. Based on these studies, the inhibitors have been approved by the FDA for treatment of advanced hormonal receptor (HR) positive breast cancers in combination with hormonal therapy. However, some evidence has recently shown unexpected effects of the inhibitors, underlining a need to characterize the effects of CDK4/6 inhibitors beyond pRb. Our study demonstrates how palbociclib impairs origin firing in the DNA replication process in pRb-deficient cell lines. Strikingly, despite the absence of pRb, cells treated with palbociclib synthesize less DNA while showing no cell cycle arrest. Furthermore, this CDK4/6 inhibitor treatment disturbs the temporal program of DNA replication and reduces the density of replication forks. Cells treated with palbociclib show a defect in the loading of the Pre-initiation complex (Pre-IC) proteins on chromatin, indicating a reduced initiation of DNA replication. Our findings highlight hidden effects of palbociclib on the dynamics of DNA replication and of its cytotoxic consequences on cell viability in the absence of pRb. This study provides a potential therapeutic application of palbociclib in combination with other drugs to target genomic instability in pRB-deficient cancers.

Cell-type-specific replication initiation programs set fragility of the FRA3B fragile site.

Common fragile sites have long been identified by cytogeneticists as chromosomal regions prone to breakage upon replication stress. They are increasingly recognized to be preferential targets for oncogene-induced DNA damage in pre-neoplastic lesions and hotspots for chromosomal rearrangements in various cancers. Common fragile site instability was attributed to the fact that they contain sequences prone to form secondary structures that may impair replication fork movement, possibly leading to fork collapse resulting in DNA breaks. Here we show, in contrast to this view, that the fragility of FRA3B--the most active common fragile site in human lymphocytes--does not rely on fork slowing or stalling but on a paucity of initiation events. Indeed, in lymphoblastoid cells, but not in fibroblasts, initiation events are excluded from a FRA3B core extending approximately 700 kilobases, which forces forks coming from flanking regions to cover long distances in order to complete replication. We also show that origins of the flanking regions fire in mid-S phase, leaving the site incompletely replicated upon fork slowing. Notably, FRA3B instability is specific to cells showing this particular initiation pattern. The fact that both origin setting and replication timing are highly plastic in mammalian cells explains the tissue specificity of common fragile site instability we observed. Thus, we propose that common fragile sites correspond to the latest initiation-poor regions to complete replication in a given cell type. For historical reasons, common fragile sites have been essentially mapped in lymphocytes. Therefore, common fragile site contribution to chromosomal rearrangements in tumours should be reassessed after mapping fragile sites in the cell type from which each tumour originates.

Stepwise activation of the ATR signaling pathway upon increasing replication stress impacts fragile site integrity.

Breaks at common fragile sites (CFS) are a recognized source of genome instability in pre-neoplastic lesions, but how such checkpoint-proficient cells escape surveillance and continue cycling is unknown. Here we show, in lymphocytes and fibroblasts, that moderate replication stresses like those inducing breaks at CFSs trigger chromatin loading of sensors and mediators of the ATR pathway but fail to activate Chk1 or p53. Consistently, we found that cells depleted of ATR, but not of Chk1, accumulate single-stranded DNA upon Mre11-dependent resection of collapsed forks. Partial activation of the pathway under moderate stress thus takes steps against fork disassembly but tolerates S-phase progression and mitotic onset. We show that fork protection by ATR is crucial to CFS integrity, specifically in the cell type where a given site displays paucity in backup replication origins. Tolerance to mitotic entry with under-replicated CFSs therefore results in chromosome breaks, providing a pool of cells committed to further instability.

Updating the mechanisms of common fragile site instability: how to reconcile the different views?

Common fragile sites (CFSs) are large chromosomal regions long identified by conventional cytogenetics as sequences prone to breakage in cells subjected to replication stress. The interest in CFSs came from their key role in the formation of DNA damage, resulting in chromosomal rearrangements. The instability of CFSs was notably correlated with the appearance of genome instability in precancerous lesions and during tumor progression. Identification of the molecular mechanisms responsible for their instability therefore represents a major challenge. A number of data show that breaks result from mitotic entry before replication completion but the mechanisms responsible for such delayed replication of CFSs and relaxed checkpoint surveillance are still debated. In addition, clues to the molecular events leading to breakage just start to emerge. We present here the results of recent reports addressing these questions.

Mistimed origin licensing and activation stabilize common fragile sites under tight DNA-replication checkpoint activation.

Genome integrity requires replication to be completed before chromosome segregation. The DNA-replication checkpoint (DRC) contributes to this coordination by inhibiting CDK1, which delays mitotic onset. Under-replication of common fragile sites (CFSs), however, escapes surveillance, resulting in mitotic chromosome breaks. Here we asked whether loose DRC activation induced by modest stresses commonly used to destabilize CFSs could explain this leakage. We found that tightening DRC activation or CDK1 inhibition stabilizes CFSs in human cells. Repli-Seq and molecular combing analyses showed a burst of replication initiations implemented in mid S-phase across a subset of late-replicating sequences, including CFSs, while the bulk genome was unaffected. CFS rescue and extra-initiations required CDC6 and CDT1 availability in S-phase, implying that CDK1 inhibition permits mistimed origin licensing and firing. In addition to delaying mitotic onset, tight DRC activation therefore supports replication completion of late origin-poor domains at risk of under-replication, two complementary roles preserving genome stability.

The Telomeric Protein TRF2 Regulates Replication Origin Activity within Pericentromeric Heterochromatin.

Heterochromatic regions render the replication process particularly difficult due to the high level of chromatin compaction and the presence of repeated DNA sequences. In humans, replication through pericentromeric heterochromatin requires the binding of a complex formed by the telomeric factor TRF2 and the helicase RTEL1 in order to relieve topological barriers blocking fork progression. Since TRF2 is known to bind the Origin Replication Complex (ORC), we hypothesized that this factor could also play a role at the replication origins (ORI) of these heterochromatin regions. By performing DNA combing analysis, we found that the ORI density is higher within pericentromeric satellite DNA repeats than within bulk genomic DNA and decreased upon TRF2 downregulation. Moreover, we showed that TRF2 and ORC2 interact in pericentromeric DNA, providing a mechanism by which TRF2 is involved in ORI activity. Altogether, our findings reveal an essential role for TRF2 in pericentromeric heterochromatin replication by regulating both replication initiation and elongation.

Transcription-mediated organization of the replication initiation program across large genes sets common fragile sites genome-wide.

Common fragile sites (CFSs) are chromosome regions prone to breakage upon replication stress known to drive chromosome rearrangements during oncogenesis. Most CFSs nest in large expressed genes, suggesting that transcription could elicit their instability; however, the underlying mechanisms remain elusive. Genome-wide replication timing analyses here show that stress-induced delayed/under-replication is the hallmark of CFSs. Extensive genome-wide analyses of nascent transcripts, replication origin positioning and fork directionality reveal that 80% of CFSs nest in large transcribed domains poor in initiation events, replicated by long-travelling forks. Forks that travel long in late S phase explains CFS replication features, whereas formation of sequence-dependent fork barriers or head-on transcription-replication conflicts do not. We further show that transcription inhibition during S phase, which suppresses transcription-replication encounters and prevents origin resetting, could not rescue CFS stability. Altogether, our results show that transcription-dependent suppression of initiation events delays replication of large gene bodies, committing them to instability.

Phosphorylation of the PCNA binding domain of the large subunit of replication factor C on Thr506 by cyclin-dependent kinases regulates binding to PCNA.

Replication factor C (RF-C) complex binds to DNA primers and loads PCNA onto DNA, thereby increasing the processivity of DNA polymerases. We have previously identified a distinct region, domain B, in the large subunit of human RF-C (RF-Cp145) which binds to PCNA. We show here that the functional interaction of RF-Cp145 with PCNA is regulated by cdk-cyclin kinases. Phosphorylation of either RF-Cp145 as a part of the RF-C complex or RF-Cp145 domain B by cdk-cyclin kinases inhibits their ability to bind PCNA. A cdk-cyclin phosphorylation site, Thr506 in RF-Cp145, identified by mass spectrometry, is also phosphorylated in vivo. A Thr506-->Ala RF-Cp145 domain B mutant is a poor in vitro substrate for cdk-cyclin kinase and, consequently, the ability of this mutant to bind PCNA was not suppressed by phosphorylation. By generating an antibody directed against phospho-Thr506 in RF-Cp145, we demonstrate that phosphorylation of endogenous RF-Cp145 at Thr506 is mediated by CDKs since it is abolished by treatment of cells with the cdk-cyclin inhibitor roscovitine. We have thus mapped an in vivo cdk-cyclin phosphorylation site within the PCNA binding domain of RF-Cp145.

Signaling from Mus81-Eme2-Dependent DNA Damage Elicited by Chk1 Deficiency Modulates Replication Fork Speed and Origin Usage.

Mammalian cells deficient in ATR or Chk1 display moderate replication fork slowing and increased initiation density, but the underlying mechanisms have remained unclear. We show that exogenous deoxyribonucleosides suppress both replication phenotypes in Chk1-deficient, but not ATR-deficient, cells. Thus, in the absence of exogenous stress, depletion of either protein impacts the replication dynamics through different mechanisms. In addition, Chk1 deficiency, but not ATR deficiency, triggers nuclease-dependent DNA damage. Avoiding damage formation through invalidation of Mus81-Eme2 and Mre11, or preventing damage signaling by turning off the ATM pathway, suppresses the replication phenotypes of Chk1-deficient cells. Damage and resulting DDR activation are therefore the cause, not the consequence, of replication dynamics modulation in these cells. Together, we identify moderate reduction of precursors available for replication as an additional outcome of DDR activation. We propose that resulting fork slowing, and subsequent firing of backup origins, helps replication to proceed along damaged templates.

USP37 deubiquitinates Cdt1 and contributes to regulate DNA replication.

DNA replication control is a key process in maintaining genomic integrity. Monitoring DNA replication initiation is particularly important as it needs to be coordinated with other cellular events and should occur only once per cell cycle. Crucial players in the initiation of DNA replication are the ORC protein complex, marking the origin of replication, and the Cdt1 and Cdc6 proteins, that license these origins to replicate by recruiting the MCM2-7 helicase. To accurately achieve its functions, Cdt1 is tightly regulated. Cdt1 levels are high from metaphase and during G1 and low in S/G2 phases of the cell cycle. This control is achieved, among other processes, by ubiquitination and proteasomal degradation. In an overexpression screen for Cdt1 deubiquitinating enzymes, we isolated USP37, to date the first ubiquitin hydrolase controlling Cdt1. USP37 overexpression stabilizes Cdt1, most likely a phosphorylated form of the protein. In contrast, USP37 knock down destabilizes Cdt1, predominantly during G1 and G1/S phases of the cell cycle. USP37 interacts with Cdt1 and is able to de-ubiquitinate Cdt1 in vivo and, USP37 is able to regulate the loading of MCM complexes onto the chromatin. In addition, downregulation of USP37 reduces DNA replication fork speed. Taken together, here we show that the deubiquitinase USP37 plays an important role in the regulation of DNA replication. Whether this is achieved via Cdt1, a central protein in this process, which we have shown to be stabilized by USP37, or via additional factors, remains to be tested.

Phosphorylation of human Fen1 by cyclin-dependent kinase modulates its role in replication fork regulation.

Cyclin-dependent kinase (Cdk) Cdk1-Cyclin A can phosphorylate Flap endonuclease 1 (Fen1), a key-enzyme of the DNA replication machinery, in late S phase. Cdk1-cyclin A forms a complex in vitro and in vivo with Fen1. Furthermore, Fen1 phosphorylation is detected in vivo and depends upon Cdks activity. As a functional consequence of phosphorylation by Cdk1-Cyclin A in vitro, endo- and exonuclease activities of Fen1 are reduced whereas its DNA binding is not affected. Moreover, phosphorylation of Fen1 by Cdk1-Cyclin A abrogates its proliferating cell nuclear antigen (PCNA) binding thus preventing stimulation of Fen1 by PCNA. Concomitantly, human cells expressing the S187A mutant defective for Cdk1-Cyclin A phosphorylation accumulate in S phase consistent with a failure in cell cycle regulation through DNA replication. Our results suggest a novel regulatory role of Cdks onto the end of S phase by targeting directly a key enzyme involved in DNA replication.

Interplay between chromatin and cell cycle checkpoints in the context of ATR/ATM-dependent checkpoints.

Maintenance of both genome stability and its structural organization into chromatin are essential to avoid aberrant gene expression that could lead to neoplasia. Genome integrity being threatened by various sources of genotoxic stresses, cells have evolved regulatory mechanisms, termed cell cycle checkpoints. In general, these surveillance pathways are thought to act mainly to coordinate proficient DNA repair with cell cycle progression. To date, this cellular response to genotoxic stress has been viewed mainly as a DNA-based signal transduction pathway. Recent studies, in both yeast and human, however, highlight possible connections between chromatin structure and cell cycle checkpoints, in particular those involving kinases of the ATM and ATR family, known as key response factors activated early in the checkpoint pathway. In this review, based on this example, we will discuss hypotheses for chromatin-based events as potential initiators of a checkpoint response or conversely, for chromatin-associated factors as targets of checkpoint proteins, promoting changes in chromatin structure, in order to make a lesion more accessible and contribute to a more efficient repair response.

TIPIN depletion leads to apoptosis in breast cancer cells.

Triple-negative breast cancer (TNBC) is the breast cancer subgroup with the most aggressive clinical behavior. Alternatives to conventional chemotherapy are required to improve the survival of TNBC patients. Gene-expression analyses for different breast cancer subtypes revealed significant overexpression of the Timeless-interacting protein (TIPIN), which is involved in the stability of DNA replication forks, in the highly proliferative associated TNBC samples. Immunohistochemistry analysis showed higher expression of TIPIN in the most proliferative and aggressive breast cancer subtypes including TNBC, and no TIPIN expression in healthy breast tissues. The depletion of TIPIN by RNA interference impairs the proliferation of both human breast cancer and non-tumorigenic cell lines. However, this effect may be specifically associated with apoptosis in breast cancer cells. TIPIN silencing results in higher levels of single-stranded DNA (ssDNA), indicative of replicative stress (RS), in TNBC compared to non-tumorigenic cells. Upon TIPIN depletion, the speed of DNA replication fork was significantly decreased in all BC cells. However, TIPIN-depleted TNBC cells are unable to fire additional replication origins in response to RS and therefore undergo apoptosis. TIPIN knockdown in TNBC cells decreases tumorigenicity in vitro and delays tumor growth in vivo. Our findings suggest that TIPIN is important for the maintenance of DNA replication and represents a potential treatment target for the worst prognosis associated breast cancers, such as TNBC.

Establishment of proliferative cell nuclear antigen gene as an internal reference gene for polymerase chain reaction of a wide range of archival and fresh mammalian tissues.

Polymerase chain reaction (PCR) from paraffin-embedded tissues provides a powerful tool to amplify DNA from a variety of recent and archival material. Because DNA from paraffin-embedded samples is more degraded than from fresh material, the amplification of reference genes is essential to exclude false-negative results. This study describes the use of the proliferative cell nuclear antigen (PCNA) gene as a reference gene in a range of animal species and in humans. The PCNA-PCR to amplify a fragment extending from exon 5 through exon 6 and including the intervening intron 6 gave a reproducible pattern, with a 280-base pair (bp) band from canine, equine, bovine, ovine, and caprine samples showing high sequence homology. Porcine, guinea pig, tiger, and lion samples, however, gave an additional fragment of approximately 197 bp. The whole intron 6 from these fragments is missing, possibly representing a pseudogene. In feline samples only the 197-bp fragment could be detected. This study shows that the PCNA gene is highly conserved across a broad range of animal species and is well suited as an internal control for PCR analysis in veterinary medicine.

The RBBP6/ZBTB38/MCM10 axis regulates DNA replication and common fragile site stability.

Faithful DNA replication is essential for the maintenance of genome integrity. Incomplete genome replication leads to DNA breaks and chromosomal rearrangements, which are causal factors in cancer and other human diseases. Despite their importance, the molecular mechanisms that control human genome stability are incompletely understood. Here, we report a pathway that is required for human genome replication and stability. This pathway has three components: an E3 ubiquitin ligase, a transcriptional repressor, and a replication protein. The E3 ubiquitin ligase RBBP6 ubiquitinates and destabilizes the transcriptional repressor ZBTB38. This repressor negatively regulates transcription and levels of the MCM10 replication factor on chromatin. Cells lacking RBBP6 experience reduced replication fork progression and increased damage at common fragile sites due to ZBTB38 accumulation and MCM10 downregulation. Our results uncover a pathway that ensures genome-wide DNA replication and chromosomal stability.

Replication dynamics: biases and robustness of DNA fiber analysis.

The factors that govern replication programs are still poorly identified in metazoans, especially in mammalian cells. Thanks to molecular combing, the dynamics of DNA replication can be assessed at the genome-scale level from the cumulative analysis of single DNA fibers. This technique notably enables measurement of replication fork speed and fork asymmetry and that of distances separating either initiation or termination events. The results presented here aim to evaluate requirements critical to accurate measurement of replication parameters by molecular combing. We show that sample size, fiber length and DNA counterstaining are crucial to gain robust information concerning replication dynamics. Our results thus provide a methodological frame to investigate the DNA replication program through molecular combing analyses.

Wee1 controls genomic stability during replication by regulating the Mus81-Eme1 endonuclease.

Correct replication of the genome and protection of its integrity are essential for cell survival. In a high-throughput screen studying H2AX phosphorylation, we identified Wee1 as a regulator of genomic stability. Wee1 down-regulation not only induced H2AX phosphorylation but also triggered a general deoxyribonucleic acid (DNA) damage response (DDR) and caused a block in DNA replication, resulting in accumulation of cells in S phase. Wee1-deficient cells showed a decrease in replication fork speed, demonstrating the involvement of Wee1 in DNA replication. Inhibiting Wee1 in cells treated with short treatment of hydroxyurea enhanced the DDR, which suggests that Wee1 specifically protects the stability of stalled replication forks. Notably, the DDR induced by depletion of Wee1 critically depends on the Mus81-Eme1 endonuclease, and we found that codepletion of Mus81 and Wee1 abrogated the S phase delay. Importantly, Wee1 and Mus81 interact in vivo, suggesting direct regulation. Altogether, these results demonstrate a novel role of Wee1 in controlling Mus81 and DNA replication in human cells.