Genome-wide experiments often measure quantitative differences between treated and untreated cells to identify affected strains. For these studies, statistical models are typically used to determine significance cutoffs. We developed a method termed “CLIK” (Cutoff Linked to Interaction Knowledge) that overlays biological knowledge from the interactome on screen results to derive a cutoff. The method takes advantage of the fact that groups of functionally related interacting genes often respond similarly to experimental conditions and, thus, cluster in a ranked list of screen results. We applied CLIK analysis to five screens of the yeast gene disruption library and found that it defined a significance cutoff that differed from traditional statistics. Importantly, verification experiments revealed that the CLIK cutoff correlated with the position in the rank order where the rate of true positives drops off significantly. In addition, the gene sets defined by CLIK analysis often provide further biological perspectives. For example, applying CLIK analysis retrospectively to a screen for cisplatin sensitivity allowed us to identify the importance of the Hrq1 helicase in DNA crosslink repair. Furthermore, we demonstrate the utility of CLIK to determine optimal treatment conditions by analyzing genome-wide screens at multiple rapamycin concentrations. We show that CLIK is an extremely useful tool for evaluating screen quality, determining screen cutoffs, and comparing results between screens. Furthermore, because CLIK uses previously annotated interaction data to determine biologically informed cutoffs, it provides additional insights into screen results, which supplement traditional statistical approaches.
Null mutations in three recombination and DNA repair genes were studied to determine their effects on mitotic recombination between the duplicate AdoMet (S-adenosylmethionine) synthetase genes (SAM1 and SAM2) in Saccharomyces cerevisiae. SAM1 and SAM2, located on chromosomes XII and IV, respectively, encode functionally equivalent although differentially regulated AdoMet synthetases. These similar but not identical (homeologous) genes are 83% homologous at the nucleotide level and this identity is limited solely to the coding regions of the genes. Single frameshift mutations were introduced into the 5' end of SAM1 and the 3' end of SAM2 by restriction site ablation. The sequences surrounding these mutations differ significantly in their degree of homology to the corresponding area of the other gene. Mitotic ectopic recombination between the mutant sam genes occurs at a rate of 8.4 x 10(-9) in a wild-type genetic background. Gene conversion of the marker within the region of greater sequence homology occurs 20-fold more frequently than conversion of the marker within the region of relative sequence diversity. The relative orientation of the two genes prevents the recovery of translocations. Mitotic recombination between the sam genes is completely dependent on the DNA repair and recombination gene RAD52. A mutation in PMS1, a mismatch repair gene, causes a 4.5-fold increase in the rate of ectopic recombination. RAD1, an excision repair gene, is required to observe this increased rate of ectopic conversion. In addition, RAD1 is involved in modulating the pattern of coconversion during recombination between the homeologous sam genes. These results suggest that interactions between mismatch repair, excision repair and recombinational repair functions are involved in determining the ectopic gene conversion frequency between the sam genes.
Cell cycle checkpoints are evolutionarily conserved surveillance systems that protect genomic stability and prevent oncogenesis in mammals. One important target of checkpoint control is ribonucleotide reductase (RNR), which catalyzes the rate-limiting step in dNTP and DNA synthesis. In both yeast and humans, RNR is transcriptionally induced after DNA damage via Mec1/Rad53 (yeast) and ATM/CHK2 (human) checkpoint pathways. In addition, yeast checkpoint proteins Mec1 and Rad53 also regulate the RNR inhibitor Sml1. After DNA damage or at S phase, Mec1 and Rad53 control the phosphorylation and concomitant degradation of Sml1 protein. This new layer of control contributes to the increased dNTP production likely necessary for DNA repair and replication; however, the molecular mechanism is unclear. Here we show that Dun1, a downstream kinase of Mec1/Rad53, genetically and physically interacts with Sml1 in vivo . The absence of Dun1 activity leads to the accumulation of Sml1 protein at S phase and after DNA damage. As a result, dun1 Δ strains need more time to finish DNA replication, are defective in mitochondrial DNA propagation, and are sensitive to DNA-damaging agents. Moreover, phospho-Sml1 is absent or dramatically reduced in dun1 Δ cells. Finally, Dun1 can phosphorylate Sml1 in vitro . These results suggest that Dun1 kinase function is the last step required in the Mec1/Rad53 cascade to remove Sml1 during S phase and after DNA damage.
We have developed an efficient method for assembling ordered cosmid contigs aligned to mega-YACs and midi-YACs (average insert sizes of 1.0 and 0.35 Mb, respectively) and used this general method to initiate high-resolution physical mapping of human chromosome 13 (Chr 13). Chr 13-enriched midi-YAC (mYAC) and mega-YAC (MYAC) sublibraries were obtained from corresponding CEPH total human YAC libraries by selecting colonies with inter-Alu PCR probes derived from Chr 13 monochromosomal cell hybrid DNA. These sublibraries were arrayed on filters at high density. In our approach, the MYAC 13 sublibrary is screened by hybridization with cytogenetically assigned Chr 13 DNA probes to select one or a small subset of MYACs. Inter-Alu PCR products from each MYAC are then hybridized to the MYAC and mYAC sublibraries to identify overlapping YACs and to an arrayed Chr 13-specific cosmid library to select corresponding cosmids. The set of selected cosmids, gridded on filters at high density, is hybridized with inter-Alu PCR products from each of the overlapping YACs to identify subsets of cosmids and also with riboprobes from each cosmid of the arrayed set ("cosmid matrix cross-hybridization"). From these data, cosmid contigs are assembled by a specifically designed computer program. Application of this method generates cosmid contigs spanning the length of a MYAC with few gaps. To provide a high-resolution map, ends of cosmids are sequenced at preselected sites to position densely spaced sequence-tagged sites.
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)
Abstract Homology-dependent exchange of genetic information between DNA molecules has a profound impact on the maintenance of genome integrity by facilitating error-free DNA repair, replication, and chromosome segregation during cell division as well as programmed cell developmental events. This chapter will focus on homologous mitotic recombination in budding yeast Saccharomyces cerevisiae. However, there is an important link between mitotic and meiotic recombination (covered in the forthcoming chapter by Hunter et al. 2015) and many of the functions are evolutionarily conserved. Here we will discuss several models that have been proposed to explain the mechanism of mitotic recombination, the genes and proteins involved in various pathways, the genetic and physical assays used to discover and study these genes, and the roles of many of these proteins inside the cell.
Saccharomyces cerevisiae cells that are mutated at TOP3, a gene that encodes a protein homologous to bacterial type I topoisomerases, have a variety of defects, including reduced growth rate, altered gene expression, blocked sporulation, and elevated rates of mitotic recombination at several loci. The rate of ectopic recombination between two unlinked, homologous loci, SAM1 and SAM2, is sixfold higher in cells containing a top3 null mutation than in wild-type cells. Mutations in either of the two other known topoisomerase genes in S. cerevisiae, TOP1 and TOP2, do not affect the rate of recombination between the SAM genes. The top3 mutation also changes the distribution of recombination events between the SAM genes, leading to the appearance of novel deletion-insertion events in which conversion tracts extend beyond the coding sequence, replacing the DNA flanking the 3' end of one SAM gene with nonhomologous DNA flanking the 3' end of the other. The effects of the top3 null mutation on recombination are dependent on the presence of an intact RAD1 excision repair gene, because both the rate of SAM ectopic gene conversion and the conversion tract length were reduced in rad1 top3 mutant cells compared with top3 mutants. These results suggest that a RAD1-dependent function is involved in the processing of damaged DNA that results from the loss of Top3 activity, targeting such DNA for repair by recombination.
