BRCA2 Regulates Transcription Elongation by RNA Polymerase II to Prevent R-Loop Accumulation

The controlled release of RNA polymerase II (RNAPII) from promoter-proximal pausing (PPP) sites is critical for transcription elongation in metazoans. We show that the human tumor suppressor BRCA2 interacts with RNAPII to regulate PPP release, thereby preventing unscheduled RNA-DNA hybrids (R-loops) implicated in genomic instability and carcinogenesis. BRCA2 inactivation by depletion or cancer-causing mutations instigates RNAPII accumulation and R-loop accrual at PPP sites in actively transcribed genes, accompanied by γH2AX formation marking DNA breakage, which is reduced by ERCC4 endonuclease depletion. BRCA2 inactivation decreases RNAPII-associated factor 1 (PAF1) recruitment (which normally promotes RNAPII release) and diminishes H2B Lys120 ubiquitination, impeding nascent RNA synthesis. PAF1 depletion phenocopies, while its overexpression ameliorates, R-loop accumulation after BRCA2 inactivation. Thus, an unrecognized role for BRCA2 in the transition from promoter-proximal pausing to productive elongation via augmented PAF1 recruitment to RNAPII is subverted by disease-causing mutations, provoking R-loop-mediated DNA breakage in BRCA2-deficient cells

Disruption of the developmentally regulated Rev3l Gene causes Embryonic Lethality

The REV3 gene encodes the catalytic subunit of DNA polymerase (pol) ζ, which can replicate past certain types of DNA lesions [1]. Saccharomyces cerevisiaerev3 mutants are viable and have lower rates of spontaneous and DNA-damage-induced mutagenesis[2]. Reduction in the level of Rev31, the presumed catalytic subunit of mammalian pol ζ, decreased damage-induced mutagenesis in human cell lines [3]. To study the function of mammalian Rev31, we inactivated the gene in mice. Two exons containing conserved DNA polymerase motifs were replaced by a cassette encoding G418 resistance and β-galactosidase, under the control of the Rev3l promoter. Surprisingly, disruption of Rev3lcaused mid-gestation embryonic lethality, with the frequency of Rev3l–/– embryos declining markedly between 9.5 and 12.5days post coitum (dpc). Rev3l–/– embryos were smaller than their heterozygous littermates and showed retarded development. Tissues in many areas were disorganised, with significantly reduced cell density. Rev3lexpression, traced by β-galactosidase staining, was first detected during early somitogenesis and gradually expanded to other tissues of mesodermal origin, including extraembryonic membranes. Embryonic death coincided with the period of more widely distributed Rev3l expression. The data demonstrate an essential function for murine Rev31 and suggest that bypass of specific types of DNAlesions by pol ζ is essential for cell viability during embryonic development in mammals