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

Members of the conserved Pif1 family of 5\u27-3\u27 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

New Topoisomerase I mutations are associated with resistance to camptothecin

<p>Abstract</p> <p>Background</p> <p>Topoisomerase I (TOP1) is a nuclear enzyme that catalyzes the relaxation of supercoiled DNA during DNA replication and transcription. TOP1 is the molecular target of camptothecin and related drugs such as irinotecan and SN38 (irinotecan's active metabolite). Irinotecan is widely used as an anti-cancer agent in the treatment of metastatic colon cancer. However, its efficacy is often limited by the development of resistance.</p> <p>Methods</p> <p>We previously established several SN38 resistant HCT116-derived clones to study the mechanisms underlying resistance to SN38. Here, we investigated whether resistance to SN38 in these cell lines could be linked to the presence of <it>TOP1 </it>mutations and changes in its expression and activity. Functional analyses were performed on these cell lines challenged with SN38 and we specifically monitored the double strands breaks with γH2AX staining and replication activity with molecular combing.</p> <p>Results</p> <p>In SN38 resistant HCT116 clones we identified three new <it>TOP1 </it>mutations, which are located in the core subdomain III (p.R621H and p.L617I) and in the linker domain (p.E710G) and are packed together at the interface between these two domains. The presence of these <it>TOP1 </it>mutations in SN38 resistant HCT116 cells did not modify TOP1 expression or intrinsic activity. Conversely, following challenge with SN38, we observed a decrease of TOP1-DNA cleavage complexes and a reduction in double-stranded break formation). In addition, we showed that SN38 resistant HCT116 cells present a strong decrease in the SN38-dependent asymmetry of replication forks that is characteristic of SN38 sensitive HCT116 cells.</p> <p>Conclusions</p> <p>These results indicate that the <it>TOP1 </it>mutations are involved in the development of SN38 resistance. We hypothesize that p.L617, p.R621 and p.E710 TOP1 residues are important for the functionality of the linker and that mutation of one of these residues is sufficient to alter or modulate its flexibility. Consequently, linker fluctuations could have an impact on SN38 binding by reducing the enzyme affinity for the drug.</p

New histone supply regulates replication fork speed and PCNA unloading

International audienc

Nucleotide-resolution DNA double-strand breaks mapping by next-generation sequencing

We present a genome-wide method to map DNA double-strand breaks (DSBs) at nucleotide resolution by direct in situ breaks labeling, enrichment on streptavidin, and next-generation sequencing (BLESS). We comprehensively validated and tested BLESS using different human and mouse cells, DSBs-inducing agents, and sequencing platforms. BLESS was able to detect telomere ends, Sce endonuclease-induced DSBs, and complex genome-wide DSBs landscapes. As a proof of principle, we characterized the genomic landscape of sensitivity to replication stress in human cells, and identified over two thousand non-uniformly distributed aphidicolin-sensitive regions (ASRs) overrepresented in genes and enriched in satellite repeats. ASRs were also enriched in regions rearranged in human cancers, with many cancer-associated genes exhibiting high sensitivity to replication stress. Our method is suitable for genome-wide mapping of DSBs in various cells and experimental conditions with a specificity and resolution unachievable by current techniques

XAB2 promotes Ku eviction from single-ended DNA double-strand breaks independently of the ATM kinase

Replication-associated single-ended DNA double-strand breaks (seDSBs) are repaired predominantly through RAD51-mediated homologous recombination (HR). Removal of the non-homologous end-joining (NHEJ) factor Ku from resected seDSB ends is crucial for HR. The coordinated actions of MRE11-CtIP nuclease activities orchestrated by ATM define one pathway for Ku eviction. Here, we identify the pre-mRNA splicing protein XAB2 as a factor required for resistance to seDSBs induced by the chemotherapeutic alkylator temozolomide. Moreover, we show that XAB2 prevents Ku retention and abortive HR at seDSBs induced by temozolomide and camptothecin, via a pathway that operates in parallel to the ATM-CtIP-MRE11 axis. Although XAB2 depletion preserved RAD51 focus formation, the resulting RAD51-ssDNA associations were unproductive, leading to increased NHEJ engagement in S/G2 and genetic instability. Overexpression of RAD51 or RAD52 rescued the XAB2 defects and XAB2 loss was synthetically lethal with RAD52 inhibition, providing potential perspectives in cancer therapy.publishedVersio

Ethanol exposure increases mutation rate through error-prone polymerases

International audienceEthanol is a ubiquitous environmental stressor that is toxic to all lifeforms. Here, we use the model eukaryote Saccharomyces cerevisiae to show that exposure to sublethal ethanol concentrations causes DNA replication stress and an increased mutation rate. Specifically, we find that ethanol slows down replication and affects localization of Mrc1, a conserved protein that helps stabilize the replisome. In addition, ethanol exposure also results in the recruitment of error-prone DNA polymerases to the replication fork. Interestingly, preventing this recruitment through mutagenesis of the PCNA/Pol30 polymerase clamp or deleting specific error-prone polymerases abolishes the mutagenic effect of ethanol. Taken together, this suggests that the mutagenic effect depends on a complex mechanism, where dysfunctional replication forks lead to recruitment of error-prone polymerases. Apart from providing a general mechanistic framework for the mutagenic effect of ethanol, our findings may also provide a route to better understand and prevent ethanol-associated carcinogenesis in higher eukaryotes

Resolution of R-loops by INO80 promotes DNA replication and maintains cancer cell proliferation and viability

Collisions between the DNA replication machinery and co-transcriptional R-loops can impede DNA synthesis and are a major source of genomic instability in cancer cells. How cancer cells deal with R-loops to proliferate is poorly understood. Here we show that the ATP-dependent chromatin remodelling INO80 complex promotes resolution of R-loops to prevent replication-associated DNA damage in cancer cells. Depletion of INO80 in prostate cancer PC3 cells leads to increased R-loops. Overexpression of the RNA:DNA endonuclease RNAse H1 rescues the DNA synthesis defects and suppresses DNA damage caused by INO80 depletion. R-loops co-localize with and promote recruitment of INO80 to chromatin. Artificial tethering of INO80 to a LacO locus enabled turnover of R-loops in cis. Finally, counteracting R-loops by INO80 promotes proliferation and averts DNA damage-induced death in cancer cells. Our work suggests that INO80-dependent resolution of R-loops promotes DNA replication in the presence of transcription, thus enabling unlimited proliferation in cancers

EXD2 Protects Stressed Replication Forks and Is Required for Cell Viability in the Absence of BRCA1/2.

Accurate DNA replication is essential to preserve genomic integrity and prevent chromosomal instability-associated diseases including cancer. Key to this process is the cells' ability to stabilize and restart stalled replication forks. Here, we show that the EXD2 nuclease is essential to this process. EXD2 recruitment to stressed forks suppresses their degradation by restraining excessive fork regression. Accordingly, EXD2 deficiency leads to fork collapse, hypersensitivity to replication inhibitors, and genomic instability. Impeding fork regression by inactivation of SMARCAL1 or removal of RECQ1's inhibition in EXD2-/- cells restores efficient fork restart and genome stability. Moreover, purified EXD2 efficiently processes substrates mimicking regressed forks. Thus, this work identifies a mechanism underpinned by EXD2's nuclease activity, by which cells balance fork regression with fork restoration to maintain genome stability. Interestingly, from a clinical perspective, we discover that EXD2's depletion is synthetic lethal with mutations in BRCA1/2, implying a non-redundant role in replication fork protection.Work in W.N.’s laboratory is funded by ICR Intramural Grant and Cancer Research UK Programme (A24881). R.A.S. and L.S. were supported by WIMM Senior Non-Clinical Fellowship awarded to W.N. M.M.S. and M.A.B. were supported by the Intramural Research Program of the NIH, National Institute on Aging, United States (Z01-AG000746-08). Work in S.G.’s laboratory is supported by BRFAA Intramural Funds. V.C. was supported by John S. Latsis Public Benefit Foundation and Alexander S. Onassis Public Benefit Foundation. Work in the P.P.’s laboratory is supported by grants from the Agence Nationale pour la Recherche (ANR), the Ligue Contre le Cancer (équipe labellisée), SIRIC Montpellier Cancer (INCa Inserm DGOS 12553), and the MSDAvenir fund

Combined inhibition of Wee1 and Chk1 as a therapeutic strategy in multiple myeloma

Multiple myeloma (MM) is a hematological malignancy characterized by an abnormal clonal proliferation of malignant plasma cells. Despite the introduction of novel agents that have significantly improved clinical outcome, most patients relapse and develop drug resistance. MM is characterized by genomic instability and a high level of replicative stress. In response to replicative and DNA damage stress, MM cells activate various DNA damage signaling pathways. In this study, we reported that high CHK1 and WEE1 expression is associated with poor outcome in independent cohorts of MM patients treated with high dose melphalan chemotherapy or anti-CD38 immunotherapy. Combined targeting of Chk1 and Wee1 demonstrates synergistic toxicities on MM cells and was associated with higher DNA double-strand break induction, as evidenced by an increased percentage of γH2AX positive cells subsequently leading to apoptosis. The therapeutic interest of Chk1/Wee1 inhibitors’ combination was validated on primary MM cells of patients. The toxicity was specific of MM cells since normal bone marrow cells were not significantly affected. Using deconvolution approach, MM patients with high CHK1 expression exhibited a significant lower percentage of NK cells whereas patients with high WEE1 expression displayed a significant higher percentage of regulatory T cells in the bone marrow. These data emphasize that MM cell adaptation to replicative stress through Wee1 and Chk1 upregulation may decrease the activation of the cell-intrinsic innate immune response. Our study suggests that association of Chk1 and Wee1 inhibitors may represent a promising therapeutic approach in high-risk MM patients characterized by high CHK1 and WEE1 expression

The BLM helicase is a new therapeutic target in multiple myeloma involved in replication stress survival and drug resistance

Multiple myeloma (MM) is a hematologic cancer characterized by accumulation of malignant plasma cells in the bone marrow. To date, no definitive cure exists for MM and resistance to current treatments is one of the major challenges of this disease. The DNA helicase BLM, whose depletion or mutation causes the cancer-prone Bloom’s syndrome (BS), is a central factor of DNA damage repair by homologous recombination (HR) and genomic stability maintenance. Using independent cohorts of MM patients, we identified that high expression of BLM is associated with a poor outcome with a significant enrichment in replication stress signature. We provide evidence that chemical inhibition of BLM by the small molecule ML216 in HMCLs (human myeloma cell lines) leads to cell cycle arrest and increases apoptosis, likely by accumulation of DNA damage. BLM inhibition synergizes with the alkylating agent melphalan to efficiently inhibit growth and promote cell death in HMCLs. Moreover, ML216 treatment re-sensitizes melphalan-resistant cell lines to this conventional therapeutic agent. Altogether, these data suggest that inhibition of BLM in combination with DNA damaging agents could be of therapeutic interest in the treatment of MM, especially in those patients with high BLM expression and/or resistance to melphalan