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A statistical approach to quantitatively derive single and double mutant fitness from colony-based growth assays is described. The resultant SGA score permits assessment of yeast fitness and genetic interactions on the genome scale. Global quantitative analysis of genetic interactions is a powerful approach for deciphering the roles of genes and mapping functional relationships among pathways. Using colony size as a proxy for fitness, we developed a method for measuring fitness-based genetic interactions from high-density arrays of yeast double mutants generated by synthetic genetic array (SGA) analysis. We identified several experimental sources of systematic variation and developed normalization strategies to obtain accurate single- and double-mutant fitness measurements, which rival the accuracy of other high-resolution studies. We applied the SGA score to examine the relationship between physical and genetic interaction networks, and we found that positive genetic interactions connect across functionally distinct protein complexes revealing a network of genetic suppression among loss-of-function alleles.

The integrity of the genome depends on diverse pathways that regulate DNA metabolism. Defects in these pathways result in genome instability, a hallmark of cancer. Deletion of ELG1 in budding yeast, when combined with hypomorphic alleles of PCNA results in spontaneous DNA damage during S phase that elicits upregulation of ribonucleotide reductase (RNR) activity. Increased RNR activity leads to a dramatic expansion of deoxyribonucleotide (dNTP) pools in G1 that allows cells to synthesize significant fractions of the genome in the presence of hydroxyurea in the subsequent S phase. Consistent with the recognized correlation between dNTP levels and spontaneous mutation, compromising ELG1 and PCNA results in a significant increase in mutation rates. Deletion of distinct genome stability genes RAD54 , RAD55 , and TSA1 also results in increased dNTP levels and mutagenesis, suggesting that this is a general phenomenon. Together, our data point to a vicious circle in which mutations in gatekeeper genes give rise to genomic instability during S phase, inducing expansion of the dNTP pool, which in turn results in high levels of spontaneous mutagenesis. Mild DNA damage checkpoint activation in yeast is sufficient to cause increased nucleotide pools and subsequent mutagenesis, revealing basal levels of replication stress as potential source of genomic instability.

BLM encodes a member of the highly conserved RecQ DNA helicase family, which is essential for the maintenance of genome stability. Homozygous inactivation of BLM gives rise to the cancer predisposition disorder Bloom's syndrome. A common feature of many RecQ helicase mutants is a hyperrecombination phenotype. In Bloom's syndrome, this phenotype manifests as an elevated frequency of sister chromatid exchanges and interhomologue recombination. We have shown previously that BLM, together with its evolutionarily conserved binding partner topoisomerase IIa (hTOPO IIIα), can process recombination intermediates that contain double Holliday junctions into noncrossover products by a mechanism termed dissolution. Here we show that a recently identified third component of the human BLM/hTOPO IIIα complex, BLAP75/ RMI1, promotes dissolution catalyzed by hTOPO IIIα. This activity of BLAP75/RMI1 is specific for dissolution catalyzed by hTOPO IIIα because it has no effect in reactions containing either Escherichia coli Top1 or Top3, both of which can also catalyze dissolution in a BLM-dependent manner. We present evidence that BLAP75/RMI1 acts by recruiting hTOPO IIIα to double Holliday junctions. Implications of the conserved ability of type IA topoisomerases to catalyze dissolution and how the evolution of factors such as BLAP75/RMI1 might confer specificity on the execution of this process are discussed.

Homologous recombination is an important mechanism for genome integrity maintenance, and several homologous recombination genes are mutated in various cancers and cancer-prone syndromes. However, since in some cases homologous recombination can lead to mutagenic outcomes, this pathway must be tightly regulated, and mitotic hyper-recombination is a hallmark of genomic instability. We performed two screens in Saccharomyces cerevisiae for genes that, when deleted, cause hyper-recombination between direct repeats. One was performed with the classical patch and replica-plating method. The other was performed with a high-throughput replica-pinning technique that was designed to detect low-frequency events. This approach allowed us to validate the high-throughput replica-pinning methodology independently of the replicative aging context in which it was developed. Furthermore, by combining the two approaches, we were able to identify and validate 35 genes whose deletion causes elevated spontaneous direct-repeat recombination. Among these are mismatch repair genes, the Sgs1-Top3-Rmi1 complex, the RNase H2 complex, genes involved in the oxidative stress response, and a number of other DNA replication, repair and recombination genes. Since several of our hits are evolutionarily conserved, and repeated elements constitute a significant fraction of mammalian genomes, our work might be relevant for understanding genome integrity maintenance in humans.

Relocalization 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 reorganization following drug-induced DNA replication stress. Changes in protein localization and abundance reveal drug-specific patterns of functional enrichments. Classification of proteins by subcellular destination enables the identification of pathways that respond to replication stress. We analysed pairwise combinations of GFP fusions and gene deletion mutants to define and order two previously unknown 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. Brown and colleagues take a systems-level approach to the DNA damage response by analysing the changes in localization and abundance of proteins in response to replication stress, using a budding yeast GFP fusion library and high-throughput microscopy.

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.

Genetic screens of the collection of ∼4500 deletion mutants in Saccharomyces cerevisiae have identified the cohort of nonessential genes that promote maintenance of genome integrity. Here we probe the role of essential genes needed for genome stability. To this end, we screened 217 tetracycline-regulated promoter alleles of essential genes and identified 47 genes whose depletion results in spontaneous DNA damage. We further showed that 92 of these 217 essential genes have a role in suppressing chromosome rearrangements. We identified a core set of 15 genes involved in DNA replication that are critical in preventing both spontaneous DNA damage and genome rearrangements. Mapping, classification, and analysis of rearrangement breakpoints indicated that yeast fragile sites, Ty retrotransposons, tRNA genes, early origins of replication, and replication termination sites are common features at breakpoints when essential replication genes that suppress chromosome rearrangements are downregulated. We propose mechanisms by which depletion of essential replication proteins can lead to double-stranded DNA breaks near these features, which are subsequently repaired by homologous recombination at repeated elements.

Oncogenesis frequently is accompanied by rampant genome instability, which fuels genetic heterogeneity and resistance to targeted cancer therapy. We have developed an approach that allows precise, quantitative measurement of genome instability in high-throughput format in the Saccharomyces cerevisiae model system. Our approach takes advantage of the strongly DNA damage-inducible gene RNR3, in conjunction with the reporter synthetic genetic array methodology, to infer mutants exhibiting genome instability by assaying for increased Rnr3 abundance. We screen for genome instability across a set of ~1000 essential and ~4200 nonessential mutant yeast alleles in untreated conditions and in the presence of the DNA-damaging agent methylmethane sulfonate. Our results provide broad insights into the cellular processes and pathways required for genome maintenance. Through comparison with existing genome instability screens, we isolated 130 genes that had not previously been linked to genome maintenance, 51% of which have human homologs. Several of these homologs are associated with a genome instability phenotype in human cells or are causally mutated in cancer. A comprehensive understanding of the processes required to prevent genome instability will facilitate a better understanding of its sources in oncogenesis.

Genome‐wide synthetic genetic interaction screens with mutants in the mus81 and mms4 replication fork‐processing genes identified a novel replication factor C (RFC) homolog, Elg1, which forms an alternative RFC complex with Rfc2–5. This complex is distinct from the DNA replication RFC, the DNA damage checkpoint RFC and the sister chromatid cohesion RFC. As expected from its genetic interactions, elg1 mutants are sensitive to DNA damage. Elg1 is redundant with Rad24 in the DNA damage response and contributes to activation of the checkpoint kinase Rad53. We find that elg1 mutants display DNA replication defects and genome instability, including increased recombination and mutation frequencies, and minichromosome maintenance defects. Mutants in elg1 show genetic interactions with pathways required for processing of stalled replication forks, and are defective in recovery from DNA damage during S phase. We propose that Elg1‐RFC functions both in normal DNA replication and in the DNA damage response.

Homologous recombination is an important mechanism for genome integrity maintenance, and several homologous recombination genes are mutated in various cancers and cancer-prone syndromes. However, since in some cases homologous recombination can lead to mutagenic outcomes, this pathway must be tightly regulated, and mitotic hyper-recombination is a hallmark of genomic instability. We performed two screens in Saccharomyces cerevisiae for genes that, when deleted, cause hyper-recombination between direct repeats. One was performed with the classical patch and replica-plating method. The other was performed with a high-throughput replica-pinning technique that was designed to detect low-frequency events. This approach allowed us to validate the high-throughput replica-pinning methodology independently of the replicative aging context in which it was developed. Furthermore, by combining the two approaches, we were able to identify and validate 35 genes whose deletion causes elevated spontaneous direct-repeat recombination. Among these are mismatch repair genes, the Sgs1-Top3-Rmi1 complex, the RNase H2 complex, genes involved in the oxidative stress response, and a number of other DNA replication, repair and recombination genes. Since several of our hits are evolutionary conserved, and repeated elements constitute a significant fraction of mammalian genomes, our work might be relevant for understanding genome integrity maintenance in humans.