Ivana Sunjevaric

Three-Dimensional Microscopy of the Rad51 Recombination Protein during Meiotic Prophase

An open question in meiosis is whether the Rad51 recombination protein functions solely in meiotic recombination or whether it is also involved in the chromosome homology search. To address this question, we have performed three-dimensional high-resolution immunofluorescence microscopy to visualize native Rad51 structures in maize male meiocytes. Maize has two closely related RAD51 genes that are expressed at low levels in differentiated tissues and at higher levels in mitotic and meiotic tissues. Cells and nuclei were specially fixed and embedded in polyacrylamide to maintain both native chromosome structure and the three dimensionality of the specimens. Analysis of Rad51 in maize meiocytes revealed that when chromosomes condense during leptotene, Rad51 is diffuse within the nucleus. Rad51 foci form on the chromosomes at the beginning of zygotene and rise to ~500 per nucleus by mid-zygotene when chromosomes are pairing and synapsing. During chromosome pairing, we consistently found two contiguous Rad51 foci on paired chromosomes. These paired foci may identify the sites where DNA sequence homology is being compared. During pachytene, the number of Rad51 foci drops to seven to 22 per nucleus. This higher number corresponds approximately to the number of chiasmata in maize meiosis. These observations are consistent with a role for Rad51 in the homology search phase of chromosome pairing in addition to its known role in meiotic recombination.

Sgs1 function in the repair of DNA replication intermediates is separable from its role in homologous recombinational repair

Mutations in human homologues of the bacterial RecQ helicase cause diseases leading to cancer predisposition and/or shortened lifespan (Werner, Bloom, and Rothmund–Thomson syndromes). The budding yeast Saccharomyces cerevisiae has one RecQ helicase, Sgs1, which functions with Top3 and Rmi1 in DNA repair. Here, we report separation‐of‐function alleles of SGS1 that suppress the slow growth of top3Δ and rmi1Δ cells similar to an SGS1 deletion, but are resistant to DNA damage similar to wild‐type SGS1. In one allele, the second acidic region is deleted, and in the other, only a single aspartic acid residue 664 is deleted. sgs1‐D664Δ, unlike sgs1Δ, neither disrupts DNA recombination nor has synthetic growth defects when combined with DNA repair mutants. However, during S phase, it accumulates replication‐associated X‐shaped structures at damaged replication forks. Furthermore, fluorescent microscopy reveals that the sgs1‐D664Δ allele exhibits increased spontaneous RPA foci, suggesting that the persistent X‐structures may contain single‐stranded DNA. Taken together, these results suggest that the Sgs1 function in repair of DNA replication intermediates can be uncoupled from its role in homologous recombinational repair.

Efficient PCR-based gene disruption in Saccharomyces strains using intergenic primers

An Erratum has been published for this article in Yeast 19(9) 2002, 803.

The Shu complex, which contains Rad51 paralogues, promotes DNA repair through inhibition of the Srs2 anti-recombinase

The Shu complex, which contains RAD51 paralogues, is involved in the decision between homologous recombination and error-prone repair. A novel role for the Shu complex in DNA recombination is proposed in which the Shu complex shifts the balance of repair toward Rad51 filament stabilization by inhibiting the disassembly reaction of Srs2.

Selective ploidy ablation, a high-throughput plasmid transfer protocol, identifies new genes affecting topoisomerase I–induced DNA damage

We have streamlined the process of transferring plasmids into any yeast strain library by developing a novel mating-based, high-throughput method called selective ploidy ablation (SPA). SPA uses a universal plasmid donor strain that contains conditional centromeres on every chromosome. The plasmid-bearing donor is mated to a recipient, followed by removal of all donor-strain chromosomes, producing a haploid strain containing the transferred plasmid. As proof of principle, we used SPA to transfer plasmids containing wild-type and mutant alleles of DNA topoisomerase I (TOP1) into the haploid yeast gene-disruption library. Overexpression of Top1 identified only one sensitive mutation, rpa34, while overexpression of top1-T(722)A allele, a camptothecin mimetic, identified 190 sensitive gene-disruption strains along with rpa34. In addition to known camptothecin-sensitive strains, this set contained mutations in genes involved in the Rpd3 histone deacetylase complex, the kinetochore, and vesicle trafficking. We further show that mutations in several ESCRT vesicle trafficking components increase Top1 levels, which is dependent on SUMO modification. These findings demonstrate the utility of the SPA technique to introduce plasmids into the haploid gene-disruption library to discover new interacting pathways.

Chromosome-scale genetic mapping using a set of 16 conditionally stable Saccharomyces cerevisiae chromosomes.

We have created a resource to rapidly map genetic traits to specific chromosomes in yeast. This mapping is done using a set of 16 yeast strains each containing a different chromosome with a conditionally functional centromere. Conditional centromere function is achieved by integration of a GAL1 promoter in cis to centromere sequences. We show that the 16 yeast chromosomes can be individually lost in diploid strains, which become hemizygous for the destabilized chromosome. Interestingly, most 2n − 1 strains endoduplicate and become 2n. We also demonstrate how chromosome loss in this set of strains can be used to map both recessive and dominant markers to specific chromosomes. In addition, we show that this method can be used to rapidly validate gene assignments from screens of strain libraries such as the yeast gene disruption collection.

Systematic hybrid LOH: a new method to reduce false positives and negatives during screening of yeast gene deletion libraries.

We have developed a new method, systematic hybrid loss of heterozygosity, to facilitate genomic screens utilizing the yeast gene deletion library. Screening is performed using hybrid diploid strains produced through mating the library haploids with strains from a different genetic background, to minimize the contribution of unpredicted recessive genetic factors present in the individual library strains. We utilize a set of strains where each contains a conditional centromere construct on one of the 16 yeast chromosomes that allows the destabilization and selectable loss of that chromosome. After mating a library gene deletion haploid to such a conditional centromere strain, which corresponds to the chromosome carrying the gene deletion, loss of heterozygosity (LOH) at the gene deletion locus can be generated in these otherwise hybrid diploids. The use of hybrid diploid strains permits complementation of any spurious recessive mutations in the library strain, facilitating attribution of the observed phenotype to the documented gene deletion and dramatically reducing false positive results commonly obtained in library screens. The systematic hybrid LOH method can be applied to virtually any screen utilizing the yeast non‐essential gene deletion library and is particularly useful for screens requiring the introduction of a genetic assay into the library strains. Copyright

Cells expressing murine RAD52 splice variants favor sister chromatid repair

ABSTRACT The RAD52 gene is essential for homologous recombination in the yeast Saccharomyces cerevisiae. RAD52 is the archetype in an epistasis group of genes essential for DNA damage repair. By catalyzing the replacement of replication protein A with Rad51 on single-stranded DNA, Rad52 likely promotes strand invasion of a double-stranded DNA molecule by single-stranded DNA. Although the sequence and in vitro functions of mammalian RAD52 are conserved with those of yeast, one difference is the presence of introns and consequent splicing of the mammalian RAD52 pre-mRNA. We identified two novel splice variants from the RAD52 gene that are expressed in adult mouse tissues. Expression of these splice variants in tissue culture cells elevates the frequency of recombination that uses a sister chromatid template. To characterize this dominant phenotype further, the RAD52 gene from the yeast Saccharomyces cerevisiae was truncated to model the mammalian splice variants. The same dominant sister chromatid recombination phenotype seen in mammalian cells was also observed in yeast. Furthermore, repair from a homologous chromatid is reduced in yeast, implying that the choice of alternative repair pathways may be controlled by these variants. In addition, a dominant DNA repair defect induced by one of the variants in yeast is suppressed by overexpression of RAD51, suggesting that the Rad51-Rad52 interaction is impaired.

The rad52-Y66A allele alters the choice of donor template during spontaneous chromosomal recombination

Spontaneous mitotic recombination is a potential source of genetic changes such as loss of heterozygosity and chromosome translocations, which may lead to genetic disease. In this study we have used a rad52 hyper-recombination mutant, rad52-Y66A, to investigate the process of spontaneous heteroallelic recombination in the yeast Saccharomyces cerevisiae. We find that spontaneous recombination has different genetic requirements, depending on whether the recombination event occurs between chromosomes or between chromosome and plasmid sequences. The hyper-recombination phenotype of the rad52-Y66A mutation is epistatic with deletion of MRE11, which is required for establishment of DNA damage-induced cohesion. Moreover, single-cell analysis of strains expressing YFP-tagged Rad52-Y66A reveals a close to wild-type frequency of focus formation, but with foci lasting 6 times longer. This result suggests that spontaneous DNA lesions that require recombinational repair occur at the same frequency in wild-type and rad52-Y66A cells, but that the recombination process is slow in rad52-Y66A cells. Taken together, we propose that the slow recombinational DNA repair in the rad52-Y66A mutant leads to a by-pass of the window-of-opportunity for sister chromatid recombination normally promoted by MRE11-dependent damage-induced cohesion thereby causing a shift towards interchromosomal recombination.

A Synthetic Dosage Lethal Genetic Interaction Between CKS1B and PLK1 Is Conserved in Yeast and Human Cancer Cells

The CKS1B gene located on chromosome 1q21 is frequently amplified in breast, lung, and liver cancers. CKS1B codes for a conserved regulatory subunit of cyclin–CDK complexes that function at multiple stages of cell cycle progression. We used a high throughput screening protocol to mimic cancer-related overexpression in a library of Saccharomyces cerevisiae mutants to identify genes whose functions become essential only when CKS1 is overexpressed, a synthetic dosage lethal (SDL) interaction. Mutations in multiple genes affecting mitotic entry and mitotic exit are highly enriched in the set of SDL interactions. The interactions between Cks1 and the mitotic entry checkpoint genes require the inhibitory activity of Swe1 on the yeast cyclin-dependent kinase (CDK), Cdc28. In addition, the SDL interactions of overexpressed CKS1 with mutations in the mitotic exit network are suppressed by modulating expression of the CDK inhibitor Sic1. Mutation of the polo-like kinase Cdc5, which functions in both the mitotic entry and mitotic exit pathways, is lethal in combination with overexpressed CKS1. Therefore we investigated the effect of targeting the human Cdc5 ortholog, PLK1, in breast cancers with various expression levels of human CKS1B. Growth inhibition by PLK1 knockdown correlates with increased CKS1B expression in published tumor cell data sets, and this correlation was confirmed using shRNAs against PLK1 in tumor cell lines. In addition, we overexpressed CKS1B in multiple cell lines and found increased sensitivity to PLK1 knockdown and PLK1 drug inhibition. Finally, combined inhibition of WEE1 and PLK1 results in less apoptosis than predicted based on an additive model of the individual inhibitors, showing an epistatic interaction and confirming a prediction of the yeast data. Thus, identification of a yeast SDL interaction uncovers conserved genetic interactions that can affect human cancer cell viability.