CRL4Wdr70 regulates H2B monoubiquitination and facilitates Exo1-dependent resection

Double strand breaks repaired by homologous recombination (HR) are first resected to form single stranded DNA which binds replication protein A (RPA). RPA attracts mediators which load the Rad51 filament to promote strand invasion, the defining feature of HR. How the resection machinery navigates nucleosome-packaged DNA is poorly understood. Using Schizosaccharomyces pombe we report that a conserved DDB1-CUL4-associated factor (DCAF), Wdr70, is recruited to DSBs as part of the Cullin4-DDB1 ubiquitin ligase (CRL4Wdr70) and stimulates distal H2B lysine 119 monoubiquitination(uH2B). Wdr70 deletion, or uH2B loss, results in increased loading of the checkpoint adaptor and resection inhibitor Crb253BP1, decreased Exo1 association and delayed resection. Wdr70 is dispensable for resection upon Crb253BP1 loss, or when the Set9 methyltransferase that creates docking sites for Crb2 is deleted. Finally we establish that this histone regulatory cascade similarly controls DSB resection in human cells

The anti-resection activity of the X protein encoded by Hepatitis Virus B

Chronic infection of hepatitis virus B (HBV) is associated with an increased incidence of hepatocellular carcinoma (HCC). HBV encodes an oncoprotein (HBx) that is crucial for viral replication and interferes with multiple cellular activities including gene expression, histone modifications and genomic stability. To date, it remains unclear how disruption of these activities contributes to hepatocarcinogenesis. Here, we report that HBV exhibits a novel anti‐resection activity by disrupting DNA end resection, thus impairing the initial steps of homologous recombination (HR). This anti‐resection activity occurs in primary human hepatocytes (PHHs) undergoing a natural viral infection‐replication cycle, as well as in cells with integrated HBV genomes. Among the seven HBV‐encoded proteins, we identified HBx as the sole viral factor that inhibits resection. By disrupting an evolutionarily conserved Cullin4A‐DDB1‐RING type of E3 ligase, CRL4WDR70, via its H‐box, we show that HBx inhibits H2B monoubiquitylation at lysine 120 (uH2B) at double strand breaks, thus reducing the efficiency of long‐range resection. We further show that directly impairing H2B monoubiquitylation elicited tumorigenesis upon engraftment of deficient cells in athymic mice, confirming that the impairment of CRL4WDR70 function by HBx is sufficient to promote carcinogenesis. Finally, we demonstrated that lack of H2B monoubiquitylation is manifest in human HBV‐associated HCC (HBVHCC) when compared with HBV‐free HCC, implying corresponding defects of epigenetic regulation and end resection. We conclude that the anti‐resection activity of HBx induces an HR defect and genome instability and contributes to tumorigenesis of host hepatocytes

Active DNA end processing in micronuclei of ovarian cancer cells

Abstract Background Ovarian cancer is one of the most deadly gynecological malignancies and inclined to recurrence and drug resistance. Previous studies showed that the tumorigenesis of ovarian cancers and their major histotypes are associated with genomic instability caused by defined sets of pathogenic mutations. In contrast, the mechanism that influences the development of drug resistance and disease recurrence is not well elucidated. Solid tumors are prone to chromosomal instability (CIN) and micronuclei formation (MN). Although MN is traditionally regarded as the outcome of genomic instability, recent investigation on its origin and final consequences reveal that the abnormal DNA metabolism in MN is a driver force for some types of catastrophic genomic rearrangements, accelerating dramatic genetic variation of cancer cells. Methods We used Indirect Immunofluorescent staining to visualize micronuclei and activation of DNA repair factors in ovarian cancer cell lines and biopsies. Results We show that ovarian cancer cells are disposed to form micronuclei upon genotoxic insults. Double strand DNA breaks (DSBs)-triggered insurgence of micronuclei is associated with unrepaired chromosomes passing through mitosis. According to their morphology and DNA staining, micronuclei compartments are divided into early and late stages that can be further characterized by differential staining of γH2AX and 53BP1. We also show that MN compartments do not halt controlled DNA metabolism as sequestered nuclear repair factors are enriched at DNA breaks in MN compartments and efficiently process DNA ends to generate single-stranded DNA (ssDNA) structures. Interestingly, unknown factors are required for DNA end processing in MN in addition to the nuclear resection machinery. Finally, these hallmarks of micronuclei evolution depicted in cell culture were recapitulated in different stages of ovarian cancer biopsies. Conclusions In aggregate, our findings demonstrate that ovarian cancer cells are inclined to form micronuclei that undergo robust DNA metabolism and generate ssDNA structures, potentially destabilizing genomic structures and triggering genetic variation

Hepatitis B virus infection disrupts homologous recombination in hepatocellular carcinoma by stabilizing resection inhibitor ADRM1

Many cancers harbor homologous recombination defects (HRDs). A HRD is a therapeutic target that is being successfully utilized in treatment of breast/ovarian cancer via synthetic lethality. However, canonical HRD caused by BRCAness mutations do not prevail in liver cancer. Here we report a subtype of HRD caused by the perturbation of a proteasome variant (CDW19S) in hepatitis B virus–bearing (HBV-bearing) cells. This amalgamate protein complex contained the 19S proteasome decorated with CRL4WDR70 ubiquitin ligase, and assembled at broken chromatin in a PSMD4Rpn10- and ATM-MDC1-RNF8–dependent manner. CDW19S promoted DNA end processing via segregated modules that promote nuclease activities of MRE11 and EXO1. Contrarily, a proteasomal component, ADRM1Rpn13, inhibited resection and was removed by CRL4WDR70-catalyzed ubiquitination upon commitment of extensive resection. HBx interfered with ADRM1Rpn13 degradation, leading to the imposition of ADRM1Rpn13-dependent resection barrier and consequent viral HRD subtype distinguishable from that caused by BRCA1 defect. Finally, we demonstrated that viral HRD in HBV-associated hepatocellular carcinoma can be exploited to restrict tumor progression. Our work clarifies the underlying mechanism of a virus-induced HRD subtype.</p