Re-localization of proteins is a hallmark of the DNA damage response. We use high-throughput microscopic screening of the yeast GFP fusion collection to develop a systems-level view of protein re-organization following drug-induced DNA replication stress. Changes in protein localization and abundance reveal drug-specific patterns of functional enrichments. Classification of proteins by sub-cellular destination allows the identification of pathways that respond to replication stress. We analyzed pairwise combinations of GFP fusions and gene deletion mutants to define and order two novel DNA damage responses. In the first, Cmr1 forms subnuclear foci that are regulated by the histone deacetylase Hos2 and are distinct from the typical Rad52 repair foci. In a second example, we find that the checkpoint kinases Mec1/Tel1 and the translation regulator Asc1 regulate P-body formation. This method identifies response pathways that were not detected in genetic and protein interaction screens, and can be readily applied to any form of chemical or genetic stress to reveal cellular response pathways.

The platinum-based drug cisplatin is a widely used first-line therapy for several cancers. Cisplatin interacts with DNA mainly in the form of Pt-d(GpG) di-adduct, which stalls cell proliferation and activates DNA damage response. Although cisplatin shows a broad spectrum of anticancer activity, its utility is limited due to acquired drug resistance and toxicity to non-targeted tissues. Here, by integrating genome-wide high-throughput Damage-seq, XR-seq, and RNA-seq approaches, along with publicly available epigenomic data, we systematically study the genome-wide profiles of cisplatin damage formation and excision repair in mouse kidney, liver, lung and spleen. We find different DNA damage and repair spectra across mouse organs, which are associated with tissue-specific transcriptomic and epigenomic profiles. The framework and the multi-omics data we present here constitute an unbiased foundation for understanding the mechanisms of cellular response to cisplatin. Our approach should be applicable for studying drug resistance and for tailoring cancer chemotherapy regimens.

In response to DNA damage, checkpoint signalling protects genome integrity at the cost of repressing cell cycle progression and DNA replication. Mechanisms for checkpoint down-regulation are therefore necessary for proper cellular proliferation. We recently uncovered a phosphatase-independent mechanism for dampening checkpoint signalling, where the checkpoint adaptor Rad9 is counteracted by the repair scaffolds Slx4-Rtt107. Here, we establish the molecular requirements for this new mode of checkpoint regulation. We engineered a minimal multi-BRCT-domain (MBD) module that recapitulates the action of Slx4-Rtt107 in checkpoint down-regulation. MBD mimics the damage-induced Dpb11-Slx4-Rtt107 complex by synergistically interacting with lesion-specific phospho-sites in Ddc1 and H2A. We propose that efficient recruitment of Dpb11-Slx4-Rtt107 or MBD via a cooperative 'two-site-docking' mechanism displaces Rad9. MBD also interacts with the Mus81 nuclease following checkpoint dampening, suggesting a spatio-temporal coordination of checkpoint signalling and DNA repair via a combinatorial mode of BRCT-domains interactions.

Obstructions to replication fork progression, referred to collectively as DNA replication stress, challenge genome stability. In Saccharomyces cerevisiae, cells lacking RTT107 or SLX4 show genome instability and sensitivity to DNA replication stress and are defective in the completion of DNA replication during recovery from replication stress. We demonstrate that Slx4 is recruited to chromatin behind stressed replication forks, in a region that is spatially distinct from that occupied by the replication machinery. Slx4 complex formation is nucleated by Mec1 phosphorylation of histone H2A, which is recognized by the constitutive Slx4 binding partner Rtt107. Slx4 is essential for recruiting the Mec1 activator Dpb11 behind stressed replication forks, and Slx4 complexes are important for full activity of Mec1. We propose that Slx4 complexes promote robust checkpoint signaling by Mec1 by stably recruiting Dpb11 within a discrete domain behind the replication fork, during DNA replication stress.

In response to replication stress, signaling mediated by DNA damage checkpoint kinases protects genome integrity. However, following repair or bypass of DNA lesions, checkpoint signaling needs to be terminated for continued cell cycle progression and proliferation. In budding yeast, the PP4 phosphatase has been shown to play a key role in preventing hyperactivation of the checkpoint kinase Rad53. In addition, we recently uncovered a phosphatase-independent mechanism for downregulating Rad53 in which the DNA repair scaffold Slx4 decreases engagement of the checkpoint adaptor Rad9 at DNA lesions. Here we reveal that proper termination of checkpoint signaling following the bypass of replication blocks imposed by alkylated DNA adducts requires the concerted action of these two fundamentally distinct mechanisms of checkpoint downregulation. Cells lacking both SLX4 and the PP4-subunit PPH3 display a synergistic increase in Rad53 signaling and are exquisitely sensitive to the DNA alkylating agent methyl methanesulfonate, which induces replication blocks and extensive formation of chromosomal linkages due to template switching mechanisms required for fork bypass. Rad53 hypersignaling in these cells seems to converge to a strong repression of Mus81-Mms4, the endonuclease complex responsible for resolving chromosomal linkages, thus explaining the selective sensitivity of slx4D pph3D cells to alkylation damage. Our results support a model in which Slx4 acts locally to downregulate Rad53 activation following fork bypass, while PP4 acts on pools of active Rad53 that have diffused from the site of lesions. We propose that the proper spatial coordination of the Slx4 scaffold and PP4 action is crucial to allow timely activation of Mus81-Mms4 and, therefore, proper chromosome segregation.

The unique nucleolar environment, the repetitive nature of ribosomal DNA (rDNA), and especially the possible involvement of RNA polymerase I (RNAPI) in transcription-coupled repair (TCR) have made the study of repair of rDNA both interesting and challenging. TCR, the transcription-dependent, preferential excision repair of the template strand compared with the nontranscribed (coding) strand has been clearly demonstrated in genes transcribed by RNAPII. Whether TCR occurs in rDNA is unresolved. In the present work, we have applied analytical methods to map repair events in rDNA using data generated by the newly developed XR-seq procedure, which measures excision repair genome-wide with single-nucleotide resolution. We find that in human and mouse cell lines, rDNA is not subject to TCR of damage caused by UV or by cisplatin.

The cohesin complex spatially organizes interphase chromatin by bringing distal genomic loci into close physical proximity, looping out the intervening DNA. Mutation of cohesin complex subunits is observed in cancer and developmental disorders, but the mechanisms through which these mutations may contribute to disease remain poorly understood. Here, we investigate a recurrent missense mutation to the hinge domain of the cohesin subunit SMC1A, observed in acute myeloid leukemia. Engineering this mutation into murine embryonic stem cells caused widespread changes in gene expression, including dysregulation of the pluripotency gene expression program. This mutation reduced cohesin levels at promoters and enhancers, decreased DNA loops and interactions across short genomic distances, and weakened insulation at CTCF-mediated DNA loops. These findings provide insight into how altered cohesin function contributes to disease and identify a requirement for the cohesin hinge domain in three-dimensional chromatin structure.

Double-strand DNA breaks occur upon exposure of cells to ionizing radiation and certain chemical agents or indirectly through replication fork collapse at DNA damage sites. If left unrepaired, double-strand breaks can cause genome instability and cell death, and their repair can result in loss of heterozygosity. In response to DNA damage, proteins involved in double-strand break repair by homologous recombination relocalize into discrete nuclear foci. We identified 29 proteins that colocalize with recombination repair protein Rad52 in response to DNA damage. Of particular interest, Ygr042w/Mte1, a protein of unknown function, showed robust colocalization with Rad52. Mte1 foci fail to form when the DNA helicase gene MPH1 is absent. Mte1 and Mph1 form a complex and are recruited to double-strand breaks in vivo in a mutually dependent manner. MTE1 is important for resolution of Rad52 foci during double-strand break repair and for suppressing break-induced replication. Together our data indicate that Mte1 functions with Mph1 in double-strand break repair.KEYWORDS DNA repair; recombination; double-strand breaks; break-induced replication; loss of heterozygosity; nuclear foci E FFECTIVE repair of double-strand DNA breaks (DSBs) is critical to the preservation of genome stability, yet most modes of DSB repair have significant potential to generate sequence alterations or sequence loss. Repair of DSBs by homologous recombination can result in loss of heterozygosity when resolution of recombination intermediates between homologous chromosomes results in a crossover. As such, cells possess several mechanisms by which crossing over can be suppressed in favor of noncrossover recombination products. Double Holliday junction (dHJ) intermediates that result from invasion of a homologous chromosome by both ends of a resected DSB (Szostak et al. 1983) can be resolved nucleolytically by the action of the Yen1 and Mus81/Mms4 endonucleases (Blanco et al. 2010;Ho et al. 2010) to produce a random distribution of crossover and noncrossover products. By contrast, the same dHJ intermediates can be dissolved by the combined helicase and ssDNA decatenase action of the Bloom/TopIIIa/Rmi1 complex (Sgs1/Top3/Rmi1 in yeast) (Wu et al. 2006;Yang et al. 2010) to yield exclusively noncrossover products (Wu and Hickson 2003). Crossovers can also be prevented if the D-loop structure that results from the first strand invasion by one end of a resected DSB into the homologous chromosome is unwound before capture of the second end to form the dHJ. Unwinding of D-loops is catalyzed in vitro and in vivo by the 39-to-59 DNA helicase Mph1 (Sun et al. 2008;Prakash et al. 2009) to prevent loss of heterozygosity due to crossovers and break-induced replication (BIR) (Luke-Glaser and Luke 2012;Mazon and Symington 2013;Stafa et al. 2014).The Mph1 DNA helicase was first identified as a deletion mutant with an increased mutation frequency (Entian et al. 1999). Subsequent characterization revealed that mph1 mutants are sensitive to the alkylating agent M...