P.H. Thorpe*, J. Bruno† and R. Rothstein* *Department of Genetics and Development, Columbia University Medical Center, New York, New York 10032; †Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Correspondence: rothstein{at}cancercenter.columbia.edu
contains Tables S1-S9. Table S1. Results of the primary yeast screen with RAS alleles RAS1 and RAS2 (wild-type) and mutants RAS1(V19) and RAS2(V19). Table S2. Results of the validation yeast screen for RAS1. Table S3. Results of the validation yeast screen for RAS2. Table S4. Results of the validation yeast screen for RAS1(V19). Table S5. Results of the validation yeast screen for RAS2(V19). Table S6. Gene deletion yeast strains sensitive to ER stress agents (Y. Chen et al., Mol Cancer Res 2005 [23]) and strains sensitive to the expression of RAS2(V19). Table S7. Gene deletion yeast strains sensitive to ERI1 deletion (M. Costanzo et al., Science 2010 [25]) and strains sensitive to the expression of RAS2(V19). Table S8. Results of the genome-wide CRISPR/Cas9 screen with the MEK inhibitor AZD6244 (selumetinib). Table S9. Results of the genome-wide CRISPR/Cas9 screen with the MEK inhibitor trametinib. (XLSX 15504 kb)
In Saccharomyces cerevisiae, MEC1 and RAD53 are essential for cell growth and checkpoint function. Their essential role in growth can be bypassed by deletion of a novel gene, SML1, which functions after several genes whose overexpression also suppresses mec1 inviability. In addition, sml1 affects various cellular processes analogous to overproducing the large subunit of ribonucleotide reductase, RNR1. These include effects on mitochondrial biogenesis, on the DNA damage response, and on cell growth. Consistent with these observations, the levels of dNTP pools in sml1Δ strains are increased compared to wild-type. This effect is not due to an increase in RNR transcription. Finally, both in vivo and in vitro experiments show that Sml1 binds to Rnr1. We propose that Sml1 inhibits dNTP synthesis posttranslationally by binding directly to Rnr1 and that Mec1 and Rad53 are required to relieve this inhibition.
The vast majority of eukaryotes possess two DNA recombinases: Rad51, which is ubiquitously expressed, and Dmc1, which is meiosis-specific. The evolutionary origins of this two-recombinase system remain poorly understood. Interestingly, Dmc1 can stabilize mismatch-containing base triplets, whereas Rad51 cannot. Here, we demonstrate that this difference can be attributed to three amino acids conserved only within the Dmc1 lineage of the Rad51/RecA family. Chimeric Rad51 mutants harboring Dmc1-specific amino acids gain the ability to stabilize heteroduplex DNA joints with mismatch-containing base triplets, whereas Dmc1 mutants with Rad51-specific amino acids lose this ability. Remarkably, RAD-51 from
DNA molecules that integrate into yeast chromosomes during yeast transformation do so by homologous recombination. We have studied the way in which circular and linear molecules recombine with homologous chromosomal sequences. We show that DNA ends are highly recombinogenic and interact directly with homologous sequences. Circular hybrid plasmids can integrate by a single reciprocal crossover, but only at a low frequency. Restriction enzyme digestion within a region homologous to yeast chromosomal DNA greatly enhances the efficiency of integration. Furthermore, if two restriction cuts are made within the same homologous sequence, thereby removing an internal segment of DNA, the resulting deleted-linear molecules are still able to transform at a high frequency. Surprisingly, the integration of these gapped-linear molecules results in replacement of the missing segment using chromosomal information. The final structure is identical to that obtained from integration of a circular molecule. The integration of linear and gapped-linear molecules, but not of circular molecules, is blocked by the rad52-1 mutation. Consideration of models for plasmid integration and gene conversion suggests that RAD52 may be involved in the DNA repair synthesis required for these processes. Implications of this work for the isolation of integrative transformants, fine-structure mapping, and the cloning of mutations are discussed.
In the yeast Saccharomyces cerevisiae , the Rad1–Rad10 protein complex participates in nucleotide excision repair (NER) and homologous recombination (HR). During HR, the Rad1–Rad10 endonuclease cleaves 3′ branches of DNA and aberrant 3′ DNA ends that are refractory to other 3′ processing enzymes. Here we show that yeast strains expressing fluorescently labeled Rad10 protein (Rad10-YFP) form foci in response to double-strand breaks (DSBs) induced by a site-specific restriction enzyme, I- Sce I or by ionizing radiation (IR). Additionally, for endonuclease-induced DSBs, Rad10-YFP localization to DSB sites depends on both RAD51 and RAD52 , but not MRE11 while IR-induced breaks do not require RAD51 . Finally, Rad10-YFP colocalizes with Rad51-CFP and with Rad52-CFP at DSB sites, indicating a temporal overlap of Rad52, Rad51 and Rad10 functions at DSBs. These observations are consistent with a putative role of Rad10 protein in excising overhanging DNA ends after homology searching and refine the potential role(s) of the Rad1–Rad10 complex in DSB repair in yeast.
The sequence of the Saccharomyces cerevisiae RAD52 gene contains five potential translation start sites and protein-blot analysis typically detects multiple Rad52 species with different electrophoretic mobilities. Here we define the gene products encoded by RAD52 . We show that the multiple Rad52 protein species are due to promiscuous choice of start codons as well as post-translational modification. Specifically, Rad52 is phosphorylated both in a cell cycle-independent and in a cell cycle-dependent manner. Furthermore, phosphorylation is dependent on the presence of the Rad52 C terminus, but not dependent on its interaction with Rad51. We also show that the Rad52 protein can be translated from the last three start sites and expression from any one of them is sufficient for spontaneous recombination and the repair of gamma-ray-induced double-strand breaks.
The yeast Mre11-Rad50-Xrs2 (MRX) complex has structural, signaling, and catalytic functions in the response to DNA damage. Xrs2, the eukaryotic-specific component of the complex, is required for nuclear import of Mre11 and Rad50 and to recruit the Tel1 kinase to damage sites. We show that nuclear-localized MR complex (Mre11-NLS) catalyzes homology-dependent repair without Xrs2, but MR cannot activate Tel1, and it fails to tether DSBs, resulting in sensitivity to genotoxins, replisome instability, and increased gross chromosome rearrangements (GCRs). Fusing the Tel1 interaction domain from Xrs2 to Mre11-NLS is sufficient to restore telomere elongation and Tel1 signaling to Xrs2-deficient cells. Furthermore, Tel1 stabilizes Mre11-DNA association, and this stabilization function becomes important for DNA damage resistance in the absence of Xrs2. Enforcing Tel1 recruitment to the nuclear MR complex fully rescues end tethering and stalled replication fork stability, and suppresses GCRs, highlighting important roles for Xrs2 and Tel1 to ensure optimal MR activity.
