DNA repair is an essential process for preserving genome integrity in all organisms. In eukaryotes, recombinational repair is choreographed by multiprotein complexes that are organized into centers (foci). Here, we analyze the cellular response to DNA double-strand breaks (DSBs) and replication stress in Saccharomyces cerevisiae. The Mre11 nuclease and the ATM-related Tel1 kinase are the first proteins detected at DSBs. Next, the Rfa1 single-strand DNA binding protein relocalizes to the break and recruits other key checkpoint proteins. Later and only in S and G2 phase, the homologous recombination machinery assembles at the site. Unlike the response to DSBs, Mre11 and recombination proteins are not recruited to hydroxyurea-stalled replication forks unless the forks collapse. The cellular response to DSBs and DNA replication stress is likely directed by the Mre11 complex detecting and processing DNA ends in conjunction with Sae2 and by RP-A recognizing single-stranded DNA and recruiting additional checkpoint and repair proteins.

Megakaryoblastic leukemia 1 (MKL1) is a myocardin-related transcription factor that we found strongly activated serum response element (SRE)-dependent reporter genes through its direct binding to serum response factor (SRF). The c-fos SRE is regulated by mitogen-activated protein kinase phosphorylation of ternary complex factor (TCF) but is also regulated by a RhoA-dependent pathway. The mechanism of this pathway is unclear. Since MKL1 (also known as MAL, BSAC, and MRTF-A) is broadly expressed, we assessed its role in serum induction of c-fos and other SRE-regulated genes with a dominant negative MKL1 mutant (DN-MKL1) and RNA interference (RNAi). We found that DN-MKL1 and RNAi specifically blocked SREdependent reporter gene activation by serum and RhoA. Complete inhibition by RNAi required the additional inhibition of the related factor MKL2 (MRTF-B), showing the redundancy of these factors. DN-MKL1 reduced the late stage of serum induction of endogenous c-fos expression, suggesting that the TCF-and RhoAdependent pathways contribute to temporally distinct phases of c-fos expression. Furthermore, serum induction of two TCF-independent SRE target genes, SRF and vinculin, was nearly completely blocked by DN-MKL1. Finally, the RBM15-MKL1 fusion protein formed by the t(1;22) translocation of acute megakaryoblastic leukemia had a markedly increased ability to activate SRE reporter genes, suggesting that its activation of SRF target genes may contribute to leukemogenesis.

Recombination is important for repairing DNA lesions, yet it can also lead to genomic rearrangements. This process must be regulated, and recently, sumoylation-mediated mechanisms were found to inhibit Rad51-dependent recombination. Here, we report that the absence of the Slx5-Slx8 complex, a newly identified player in the SUMO (small ubiquitin-like modifier) pathway, led to increased Rad51-dependent and Rad51-independent recombination. The increases were most striking during S phase, suggesting an accumulation of DNA lesions during replication. Consistent with this view, Slx8 protein localized to replication centers. In addition, like SUMO E2 mutants, slx8⌬ mutants exhibited clonal lethality, which was due to the overamplification of 2m, an extrachromosomal plasmid. Interestingly, in both SUMO E2 and slx8⌬ mutants, clonal lethality was rescued by deleting genes required for Rad51-independent recombination but not those involved in Rad51-dependent events. These results suggest that sumoylation negatively regulates Rad51-independent recombination, and indeed, the Slx5-Slx8 complex affected the sumoylation of several enzymes involved in early steps of Rad51-independent recombination. We propose that, during replication, the Slx5-Slx8 complex helps prevent DNA lesions that are acted upon by recombination. In addition, the complex inhibits Rad51-independent recombination via modulating the sumoylation of DNA repair proteins.The maintenance of genome stability is critical for cell survival and for the proper development of an organism. It requires a network of genes that must be coordinated during various DNA metabolic processes. In the budding yeast Saccharomyces cerevisiae, the SLX5 (or HEX3) and SLX8 genes are among the guardians of genomic stability. Originally identified as genes required for the viability of cells lacking Sgs1 (the homolog of human BLM and WRN), both SLX5 and SLX8 were subsequently shown to be required for the viability or fitness of many other strains with mutations that affect genomic integrity (29,31,40). Particularly, these genes exhibit extensive interactions with genes involved in replication or replication fork stability, such as RAD27, POL32, ELG1, and DBF2, suggesting a role for these genes in replication and/or repair (31). Consistent with this view, the deletion of SLX5 or SLX8 leads to a 150-to 200-fold increase in gross chromosomal rearrangement and a 4-fold increase in spontaneous mutation rates (48). These findings point to the importance of Slx5 and Slx8 in the maintenance of genome stability.Based on biochemical and genetic evidence, Slx5 and Slx8 proteins function as a complex (29,47,48). Several recent studies suggest that the Slx5-Slx8 complex participates in the sumoylation pathway, which entails the addition of a small ubiquitin-like modifier (SUMO) to the target proteins. Sumoylation requires the sequential action of E1, E2, and E3 enzymes; while only a single E1 and a single E2 exist in previously studied organisms, multiple E3s have been found and are thought to confer ...

Summary The DNA damage response (DDR) occurs in the context of chromatin structure, and architectural features of chromatin contribute to DNA damage signaling and repair. While the role of chromatin decondensation in the DDR is established, we show here that chromatin condensation is integral to DDR signaling. We find that upon DNA damage, chromatin regions transiently expand before undergoing extensive compaction. Using a protein-chromatin tethering system to create defined chromatin domains, we show that interference with chromatin condensation results in failure to fully activate DDR. Conversely, forced induction of local chromatin condensation promotes ATM- and ATR-dependent activation of upstream DDR signaling in a break-independent manner. Finally, while persistent chromatin compaction enhanced upstream DDR signaling from irradiation-induced breaks, it reduced recovery and survival after damage. Our results demonstrate that chromatin condensation is sufficient for activation of DDR signaling and is an integral part of physiological DDR signaling.