A Genome-Wide Screen for Genes Affecting Spontaneous Direct-Repeat Recombination inSaccharomyces cerevisiae

Abstract

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.

High-throughput replica-pinning approach to screen for yeast genes controlling low-frequency events

“…This protocol describes a high-throughput replica pinning procedure to screen for genes affecting low-frequency events using an appropriate selectable genetic reporter ( Figure 1 ). We have successfully applied this protocol to study the accumulation of spontaneous mutations during replicative aging using inactivation of the Mother Enrichment Program ( Lindstrom and Gottschling, 2009 ) as a readout for spontaneous mutagenesis events ( Novarina et al., 2020a ), and to screen for genes that affect spontaneous direct-repeat recombination with the leu2ΔEcoRI-URA3-leu2ΔBstEII direct-repeat recombination reporter ( Novarina et al., 2020b ; Smith and Rothstein, 1999 ). As a standard example, we describe here the procedure for the spontaneous direct-repeat recombination screen ( Novarina et al., 2020b ).…”

“…We have successfully applied this protocol to study the accumulation of spontaneous mutations during replicative aging using inactivation of the Mother Enrichment Program ( Lindstrom and Gottschling, 2009 ) as a readout for spontaneous mutagenesis events ( Novarina et al., 2020a ), and to screen for genes that affect spontaneous direct-repeat recombination with the leu2ΔEcoRI-URA3-leu2ΔBstEII direct-repeat recombination reporter ( Novarina et al., 2020b ; Smith and Rothstein, 1999 ). As a standard example, we describe here the procedure for the spontaneous direct-repeat recombination screen ( Novarina et al., 2020b ). The protocol can be easily adapted to test other forms of genomic instability, or other low-frequency events, such as gross chromosomal rearrangements ( Schmidt et al., 2006 ), inverted-repeat recombination ( Rattray and Symington, 1994 ), transcription errors ( Irvin et al., 2014 ; Strathern et al., 2012 ), transient loss of gene silencing ( Dodson and Rine, 2015 ) and read-through at premature termination codons ( Altamura et al., 2016 ), provided a genetically selectable reporter is available or could be developed.…”

“…This approach can be used to screen genes that control any process involving low-frequency events for which genetically selectable reporters are available, e.g., spontaneous mutations, recombination, and transcription errors. For complete details on the use and execution of this protocol, please refer to ( Novarina et al., 2020a , 2020b ).…”

“…For complete details on the use and execution of this protocol, please refer to ( Novarina et al., 2020a , 2020b ).…”

A case of convergent-gene interference in the budding yeast knockout library causing chromosome instability

“…One such gene that has consistently appeared in budding yeast screens related to altered CIN as well as genome instability is SNO1 ( Novarina et al 2020 ; Stirling et al 2011 ; Yuen et al 2007 ). Sno1 is annotated as a protein of uncharacterized function on the Saccharomyces Genome Database (SGD) with a predicted function in the pyridoxine biosynthesis pathway although its glutaminase activity has only been demonstrated in vitro ( Dong et al 2004 ; Padilla et al 1998 ).…”