Chromosomal double-strand breaks (DSBs) are resected by 5′-nucleases to form 3′ single-strand DNA (ssDNA) substrates for binding by homologous recombination and DNA damage checkpoint proteins. Two redundant pathways of extensive resection were described both in cells 1-3 and in vitro 4-6, one relying on Exo1 exonuclease and the other on Sgs1 helicase and Dna2 nuclease. However, it remains unknown how resection proceeds within the context of chromatin where histones and histone-bound proteins represent barriers for resection enzymes. Here, we have identified the yeast nucleosome remodeling enzyme Fun30 as novel factor promoting DSB end resection. Fun30 is the major nucleosome remodeler promoting extensive Exo1- and Sgs1-dependent resection of DSBs while the RSC and INO80 chromatin remodeling complexes play redundant roles with Fun30 in resection adjacent to DSB ends. ATPase and helicase domains of Fun30, which are needed for nucleosome remodeling 7, are also required for resection. Fun30 is robustly recruited to DNA breaks and spreads around the DSB coincident with resection. Fun30 becomes less important for resection in the absence of the histone-bound Rad9 checkpoint adaptor protein known to block 5′ strand processing 8 and in the absence of either histone H3 K79 methylation or γ-H2A, which mediate recruitment of the Rad9 9, 10. Together these data suggest that Fun30 helps to overcome the inhibitory effect of Rad9 on DNA resection.

Comprehensive discovery of genetic mechanisms of drug resistance and identification of in vivo drug targets represent significant challenges. Here we present a functional variomics technology in the model organism Saccharomyces cerevisiae. This tool analyzes numerous genetic variants and effectively tackles both problems simultaneously. Using this tool, we discovered almost all genes that, due to mutations or modest overexpression, confer resistance to rapamycin, cycloheximide, and amphotericin B. Most significant among the resistance genes were drug targets, including multiple targets of a given drug. With amphotericin B, we discovered the highly conserved membrane protein Pmp3 as a potent resistance factor and a possible novel target. Widespread application of this tool should allow rapid identification of conserved resistance mechanisms and targets of many more compounds. New genes and alleles that confer resistance to other stresses can also be discovered. Similar tools in other systems such as human cell lines will also be useful.

DNA double-strand breaks (DSBs) are one of the most cytotoxic types of DNA lesion challenging genome integrity. The activity of cyclin-dependent kinase Cdk1 is essential for DSB repair by homologous recombination and for DNA damage signaling. Here we identify the Fun30 chromatin remodeler as a new target of Cdk1. Fun30 is phosphorylated by Cdk1 on Serine 28 to stimulate its functions in DNA damage response including resection of DSB ends. Importantly, Cdk1-dependent phosphorylation of Fun30-S28 increases upon DNA damage and requires the recruitment of Fun30 to DSBs, suggesting that phosphorylation increases in situ at the DNA damage. Consistently, we find that Cdk1 and multiple cyclins become highly enriched at DSBs and that the recruitment of Cdk1 and cyclins Clb2 and Clb5 ensures optimal Fun30 phosphorylation and checkpoint activation. We propose that the enrichment of Cdk1-cyclin complexes at DSBs serves as a mechanism for enhanced targeting and modulating of the activity of DNA damage response proteins.

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