Genotoxic treatments in human cells consistently induce uncoupling of replication forks and their remodeling into four-way junctions by the RAD51 recombinase.

Besides its role in homologous recombination, the tumor suppressor BRCA2 protects stalled replication forks from nucleolytic degradation. Defective fork stability contributes to chemotherapeutic sensitivity of BRCA2-defective tumors by yet-elusive mechanisms. Using DNA fiber spreading and direct visualization of replication intermediates, we report that reversed replication forks are entry points for fork degradation in BRCA2-defective cells. Besides MRE11 and PTIP, we show that RAD52 promotes stalled fork degradation and chromosomal breakage in BRCA2-defective cells. Inactivation of these factors restores reversed fork frequency and chromosome integrity in BRCA2-defective cells. Conversely, impairing fork reversal prevents fork degradation, but increases chromosomal breakage, uncoupling fork protection, and chromosome stability. We propose that BRCA2 is dispensable for RAD51-mediated fork reversal, but assembles stable RAD51 nucleofilaments on regressed arms, to protect them from degradation. Our data uncover the physiopathological relevance of fork reversal and illuminate a complex interplay of homologous recombination factors in fork remodeling and stability.

SummaryConcomitant hepatocyte apoptosis and regeneration is a hallmark of chronic liver diseases (CLDs) predisposing to hepatocellular carcinoma (HCC). Here, we mechanistically link caspase-8-dependent apoptosis to HCC development via proliferation- and replication-associated DNA damage. Proliferation-associated replication stress, DNA damage, and genetic instability are detectable in CLDs before any neoplastic changes occur. Accumulated levels of hepatocyte apoptosis determine and predict subsequent hepatocarcinogenesis. Proliferation-associated DNA damage is sensed by a complex comprising caspase-8, FADD, c-FLIP, and a kinase-dependent function of RIPK1. This platform requires a non-apoptotic function of caspase-8, but no caspase-3 or caspase-8 cleavage. It may represent a DNA damage-sensing mechanism in hepatocytes that can act via JNK and subsequent phosphorylation of the histone variant H2AX.

SummaryInterstrand cross-links (ICLs) are toxic DNA lesions interfering with DNA metabolism that are induced by widely used anticancer drugs. They have long been considered absolute roadblocks for replication forks, implicating complex DNA repair processes at stalled or converging replication forks. Recent evidence challenged this view, proposing that single forks traverse ICLs by yet elusive mechanisms. Combining ICL immunolabeling and single-molecule approaches in human cells, we now show that ICL induction leads to global replication fork slowing, involving forks not directly challenged by ICLs. Active fork slowing is linked to rapid recruitment of RAD51 to replicating chromatin and to RAD51/ZRANB3-mediated fork reversal. This global modulation of fork speed and architecture requires ATR activation, promotes single-fork ICL traverse—here, directly visualized by electron microscopy—and prevents chromosomal breakage by untimely ICL processing. We propose that global fork slowing by remodeling provides more time for template repair and promotes bypass of residual lesions, limiting fork-associated processing.

Homologous recombination (HR) is a key pathway that repairs DNA double-strand breaks (DSBs) and helps to restart stalled or collapsed replication forks. How HR supports replication upon genotoxic stress is not understood. Using in vivo and in vitro approaches, we show that the MMS22L-TONSL heterodimer localizes to replication forks under unperturbed conditions and its recruitment is increased during replication stress in human cells. MMS22L-TONSL associates with replication protein A (RPA)-coated ssDNA, and the MMS22L subunit directly interacts with the strand exchange protein RAD51. MMS22L is required for proper RAD51 assembly at DNA damage sites in vivo, and HR-mediated repair of stalled forks is abrogated in cells expressing a MMS22L mutant deficient in RAD51 interaction. Similar to the recombination mediator BRCA2, recombinant MMS22L-TONSL limits the assembly of RAD51 on dsDNA, which stimulates RAD51-ssDNA nucleoprotein filament formation and RAD51-dependent strand exchange activity in vitro. Thus, by specifically regulating RAD51 activity at uncoupled replication forks, MMS22L-TONSL stabilizes perturbed replication forks by promoting replication fork reversal and stimulating their HR-mediated restart in vivo.

Esophageal cancer incidence is increasing and has few treatment options. In studying receptor tyrosine kinases associated with esophageal cancers, we have identified EPHB4 to be robustly overexpressed in cell lines and primary tumor tissues. In total, 94 squamous cell carcinoma, 82 adenocarcinoma, 25 dysplasia, 13 Barrett esophagus, and 25 adjacent or unrelated normal esophageal tissues were evaluated by immunohistochemistry. EPHB4 expression was significantly higher in all the different histologic categories than in adjacent normal tissues. In 13 esophageal cancer cell lines, 3 of the 9 SCC cell lines and 2 of the 4 adenocarcinomas expressed very high levels of EPHB4. An increased gene copy number ranging from 4 to 20 copies was identified in a subset of the overexpressing patient samples and cell lines. We have developed a novel 4-nitroquinoline 1-oxide (4-NQO)-induced mouse model of esophageal cancer that recapitulates the EPHB4 expression in humans. A specific small-molecule inhibitor of EPHB4 decreased cell viability in a timeand dose-dependent manner in 3 of the 4 cell lines tested. The small-molecule inhibitor and an EPHB4 siRNA also decreased cell migration (12%-40% closure in treated vs. 60%-80% in untreated), with decreased phosphorylation of various tyrosyl-containing proteins, EphB4, and its downstream target p125FAK. Finally, in a xenograft tumor model, an EPHB4 inhibitor abrogated tumor growth by approximately 60% compared with untreated control. EphB4 is robustly expressed and potentially serves as a novel biomarker for targeted therapy in esophageal cancers. Cancer Res; 73(1); 184-94. Ó2012 AACR.

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DNA interstrand crosslinks (ICLs) induced by endogenous aldehydes or chemotherapeutic agents interfere with essential processes such as replication and transcription. ICL recognition and repair by the Fanconi Anemia pathway require the formation of an X-shaped DNA structure that may arise from convergence of two replication forks at the crosslink or traversing of the lesion by a single replication fork. Here, we report that ICL traverse strictly requires DNA repriming events downstream of the lesion, which are carried out by PrimPol, the second primase-polymerase identified in mammalian cells after Pola/Primase. The recruitment of PrimPol to the vicinity of ICLs depends on its interaction with RPA, but not on FANCM translocase or the BLM/TOP3A/RMI1-2 (BTR) complex that also participate in ICL traverse. Genetic ablation of PRIMPOL makes cells more dependent on the fork convergence mechanism to initiate ICL repair, and PRIMPOL KO cells and mice display hypersensitivity to ICL-inducing drugs. These results open the possibility of targeting PrimPol activity to enhance the efficacy of chemotherapy based on DNA crosslinking agents.