After UV irradiation, DNA polymerases specialized in translesion DNA synthesis (TLS) aid DNA replication. However, it is unclear whether other mechanisms also facilitate the elongation of UVdamaged DNA. We wondered if Rad51 recombinase (Rad51), a factor that escorts replication forks, aids replication across UV lesions. We found that depletion of Rad51 impairs S-phase progression and increases cell death after UV irradiation. Interestingly, Rad51 and the TLS polymerase polη modulate the elongation of nascent DNA in different ways, suggesting that DNA elongation after UV irradiation does not exclusively rely on TLS events. In particular, Rad51 protects the DNA synthesized immediately before UV irradiation from degradation and avoids excessive elongation of nascent DNA after UV irradiation. In Rad51-depleted samples, the degradation of DNA was limited to the first minutes after UV irradiation and required the exonuclease activity of the double strand break repair nuclease (Mre11). The persistent dysregulation of nascent DNA elongation after Rad51 knockdown required Mre11, but not its exonuclease activity, and PrimPol, a DNA polymerase with primase activity. By showing a crucial contribution of Rad51 to the synthesis of nascent DNA, our results reveal an unanticipated complexity in the regulation of DNA elongation across UV-damaged templates.PrimPol | polκ | polη | DNA damage tolerance | DNA replication T he DNA-binding protein Rad51 is a central component of homologous recombination repair (HRR). HRR repairs doublestrand breaks (DSBs) in an error-free way and processes one-ended DSBs to reactivate collapsed replication forks (1). During HRR, DSBs are processed by the 3′-to-5′ exonuclease activity of the double strand break repair nuclease (Mre11) to generate protruding 3′ ssDNA at DSBs. The ssDNA is then coated with Rad51, a factor that catalyzes homology search and strand invasion. The loading and stabilization of Rad51/ssDNA complexes are supported by multiple mediators, such as the tumor suppressor BRCA2 (breast cancer 2) (1). Moreover, Rad51 promotes XPF1-and Exo1-mediated DSB formation after gemcitabine-induced irreversible ribonucleotide reductase inhibition, thus promoting cell death (2). The signals that redirect Rad51 into a DSB formation pathway rather than DSB repair are not yet known.The functions of Rad51 are not limited to the processing/ generation of DSBs. Over the past few years, it has become evident that Rad51 escorts ongoing replication forks regardless of the presence of DSBs (3-5). Specifically, Rad51 protects persistently stalled replication forks from Mre11-mediated nucleolytic degradation and facilitates replication fork restart when the replication-halting agent hydroxyurea (HU) or aphidicolin (APH) is removed (6-19). Such novel functions of Rad51 require many HRR factors, including BCRA2, FANCD2 (Fanconi Anemia Complementation group protein D2), CtIP, BRCA1, and the WRN helicase, but are independent of HRR effectors, such as Rad54 (6, 7). Rad51-dependent fork-restart and fork-protection ...

Hypoxia-inducible factors (HIFs) are a family of evolutionary conserved alpha-beta heterodimeric transcription factors that induce a wide range of genes in response to low oxygen tension. Molecular mechanisms that mediate oxygen-dependent HIF regulation operate at the level of the alpha subunit, controlling protein stability, subcellular localization, and transcriptional coactivator recruitment. We have conducted an unbiased genome-wide RNA interference (RNAi) screen in Drosophila cells aimed to the identification of genes required for HIF activity. After 3 rounds of selection, 30 genes emerged as critical HIF regulators in hypoxia, most of which had not been previously associated with HIF biology. The list of genes includes components of chromatin remodeling complexes, transcription elongation factors, and translational regulators. One remarkable hit was the argonaute 1 (ago1) gene, a central element of the microRNA (miRNA) translational silencing machinery. Further studies confirmed the physiological role of the miRNA machinery in HIF–dependent transcription. This study reveals the occurrence of novel mechanisms of HIF regulation, which might contribute to developing novel strategies for therapeutic intervention of HIF–related pathologies, including heart attack, cancer, and stroke.