Development of a novel screen protocol for the identification of genes causing replication associated genomic instability in Schizosaccharomyces pombe

Abstract

Replication fork stalling is a source of potentially tumourigenic genomic instability. The RecQ family helicase, Rqh1, is critical for the prevention of replication fork collapse and the formation of potentially deleterious recombination intermediates following fork stalling. Previous work in our lab with Schizosaccharomyces pombe (fission yeast) has shown that rqh10/rqh10 diploids are inherently unstable and show rapid reversion to the haploid state. The current work exploits this characteristic of fission yeast rqh10 mutants in a screen for genes that normally promote replication associated genomic instability. The rqh10rad30 mutant strains employed in this work incorporate the checkpoint deficiency caused by a lack of Rad3, so as to exacerbate the genomically unstable nature of this model. The current work describes the lithium acetate transformation based random mutagenesis by non-homologous integration of the ura4+ selectable marker into the rqh10rad30 fission yeast strains. This random mutagenesis generated extensive (24,500 – 50,000) mutant libraries. The quality of the libraries was assessed by can1 mutant assay, confirming an adequately extensive mutagenesis for the proposed screen. The process to be employed in the screen would involve the crossing of the mutant libraries, with the hope of generating diploids that will have two mutant copies of the same gene. Some of these diploids would appear unusually stable, showing a normal sporulation phenotype. This would indicate the mutation of a gene that normally promotes genomic instability following replication fork stalling. The practicality of the proposed screen of a vast number of diploids was assessed and described in detail in the current work. A technique involving inverse PCR (IPCR) adopted from previous work to identify mutants of interest, was also investigated. The investigation of this technique, and the work of others, suggests that transformation using such selectable marker fragments results in most apparent transformants containing extrachromosomal ura4+ fragments. These fragments are thought to provide the predominant template for IPCR, rendering the process unsuccessful at identifying the mutation in the current screen. However, with the mutant libraries generated, and the screen procedure detailed, the stage is set to conduct the screen once a more appropriate mutation location technique is identified.