Summary PRP19 is a ubiquitin ligase involved in pre-mRNA splicing and the DNA damage response (DDR). While the role for PRP19 in splicing is well characterized, its role in the DDR remains elusive. Through a proteomic screen for proteins that interact with RPA-coated single-stranded DNA (RPA-ssDNA), we identified PRP19 as a sensor of DNA damage. PRP19 binds RPA directly and localizes to DNA damage sites via RPA, promoting RPA ubiquitylation in a DNA damage-induced manner. PRP19 facilitates the accumulation of ATRIP, the regulatory partner of the ATR kinase, at DNA damage sites. Depletion of PRP19 compromised the phosphorylation of ATR substrates, the recovery of stalled replication forks, and the progression of replication forks on damaged DNA. Importantly, PRP19 mutants that cannot bind RPA or function as an E3 ligase failed to support the ATR response, revealing that PRP19 drives ATR activation by acting as an RPA-ssDNA-sensing ubiquitin ligase during the DDR.

Eukaryotic DNA replication produces sister chromatids that are linked together until anaphase by cohesin, a ring-shaped protein complex that is thought to act by embracing both chromatids. Cohesin is enriched at centromeres, as well as discrete sites along chromosome arms where transcription positions the complex between convergent gene pairs. A relationship between cohesin and Sir-mediated transcriptional silencing has also begun to emerge. Here we used fluorescence microscopy and site-specific recombination to characterize interactions between newly replicated copies of the silent HMR mating-type locus. HMR was tagged with lac-GFP and flanked by binding sites for an inducible site-specific recombinase. Excision of the locus in cells with sister chromatids produced two chromatin circles that remained associated with one another. Pairing of the circles required silent chromatin, cohesin, and the RSC chromatin-remodeling complex. Chromatin immunoprecipitation showed that targeting of cohesin to the locus is Sir-dependent, and functional tests showed that silent chromatin acts in a continuous fashion to maintain cohesion. Remarkably, loss of silencing led to loss of cohesin from linear chromosomal templates but not from excised chromatin circles. The results are consistent with a model in which cohesin binds silent chromatin via topological linkage to individual chromatids.[Keywords: Sir; transcriptional silencing; silent chromatin; sister chromatid cohesion; cohesin; RSC] Supplemental material is available at http://www.genesdev.org. Faithful segregation of chromosomes between dividing cells relies on sister chromatid cohesion, a process by which newly replicated sister chromatids adhere to one another until they align on the bipolar metaphase spindle (Nasmyth 2001;Uhlmann 2004). When all chromosome pairs attach to microtubules from both spindle poles (biorientation), cohesion of sister chromatids is destroyed and anaphase separation commences. Cohesion is reestablished in the next cell cycle as chromosomes are duplicated during S phase. Sister chromatid cohesion relies on a multisubunit complex, termed cohesin, that consists of a heterodimer of SMC proteins Smc1 and Smc3, as well as two non-SMC proteins, Scc3 and Scc1/ Mcd1 (hereafter referred to as Scc1). The proteins are thought to form a ring-shaped complex with a central hole large enough for two nucleosomal fibers. According to one compelling model, cohesin embraces both sister chromatids to counter the opposing force of spindle microtubules (Gruber et al. 2003). In a competing model, sisters are "snapped" together by interacting pairs of cohesin complexes (Milutinovich and Koshland 2003). In either case, destruction of cohesin at anaphase is triggered by separase, a site-specific protease that cleaves Scc1.Cohesin has been mapped to centromeres and discrete sites along chromosomal arms in both budding and fission yeasts (Blat and Kleckner 1999;Tanaka et al. 1999;Laloraya et al. 2000;Glynn et al. 2004;Lengronne et al. 2004). Rules governing binding specific...

The ATR (ATM [ataxia telangiectasia-mutated]-and Rad3-related) checkpoint is a crucial DNA damage signaling pathway. While the ATR pathway is known to transmit DNA damage signals through the ATR-Chk1 kinase cascade, whether post-translational modifications other than phosphorylation are important for this pathway remains largely unknown. Here, we show that protein SUMOylation plays a key role in the ATR pathway. ATRIP, the regulatory partner of ATR, is modified by SUMO2/3 at K234 and K289. An ATRIP mutant lacking the SUMOylation sites fails to localize to DNA damage and support ATR activation efficiently. Surprisingly, the ATRIP SUMOylation mutant is compromised in the interaction with a protein group, rather than a single protein, in the ATR pathway. Multiple ATRIP-interacting proteins, including ATR, RPA70, TopBP1, and the MRE11-RAD50-NBS1 complex, exhibit reduced binding to the ATRIP SUMOylation mutant in cells and display affinity for SUMO2 chains in vitro, suggesting that they bind not only ATRIP but also SUMO. Fusion of a SUMO2 chain to the ATRIP SUMOylation mutant enhances its interaction with the protein group and partially suppresses its localization and functional defects, revealing that ATRIP SUMOylation promotes ATR activation by providing a unique type of protein glue that boosts multiple protein interactions along the ATR pathway.[Keywords: ATR; ATRIP; checkpoint; SUMOylation] Supplemental material is available for this article.Received January 18, 2014; revised version accepted June 2, 2014.The maintenance of genomic stability requires not only DNA repair machineries but also signal transduction pathways that regulate and coordinate the DNA damage response (DDR) (Ciccia and Elledge 2010). In human cells, DNA damage signaling is primarily initiated by the ataxia telangiectasia-mutated (ATM) and the ATMand Rad3-related (ATR) kinases. Whereas ATM is activated by DNA double-stranded breaks (DSBs), ATR is elicited by a much broader spectrum of DNA damage and replication stress (Cimprich and Cortez 2008;Marechal and Zou 2013;Shiloh and Ziv 2013). Once activated, ATM and ATR phosphorylate and activate their effector kinases, Chk2 and Chk1, respectively. Together, the ATMChk2 and ATR-Chk1 kinase cascades phosphorylate a number of substrates involved in DNA repair, DNA replication, and cell cycle transitions, coordinating these processes to suppress genomic instability. In addition to the phosphorylation events mediated by the ATM and ATR pathways, several other types of post-translational modifications (PTMs), such as ubiquitylation, SUMOylation, methylation, acetylation, and poly-ADP ribosylation, are also implicated in the DDR (Bekker-Jensen and Mailand 2010;Huen et al. 2010;Lukas et al. 2011;Jackson and Durocher 2013). We recently found that the efficient activation of ATR relies on a ubiquitylation circuitry mediated by RPA-ssDNA (RPA-coated ssDNA) and PRP19 (Marechal et al. 2014). This new finding raises a question as to whether other PTMs also participate in DNA damage signaling through the ATR path...