Comprehensive Synthetic Genetic Array Analysis of Alleles That Interact with Mutation of the Saccharomyces cerevisiae RecQ Helicases Hrq1 and Sgs1

Abstract

Most eukaryotic genomes encode multiple RecQ family helicases, including five such enzymes in humans. For many years, the yeast Saccharomyces cerevisiae was considered unusual in that it only contained a single RecQ helicase, named Sgs1. However, it has recently been discovered that a second RecQ helicase, called Hrq1, resides in yeast. Both Hrq1 and Sgs1 are involved in genome integrity, functioning in processes such as DNA inter-strand crosslink repair, double-strand break repair, and telomere maintenance. However, it is unknown if these enzymes interact at a genetic, physical, or functional level as demonstrated for their human homologs. Thus, we performed synthetic genetic array (SGA) analyses of hrq1D and sgs1D mutants. As inactive alleles of helicases can demonstrate dominant phenotypes, we also performed SGA analyses on the hrq1-K318A and sgs1-K706A ATPase/helicase-null mutants, as well as all combinations of deletion and inactive double mutants. We crossed these eight query strains (hrq1D, sgs1D, hrq1-K318A, sgs1-K706A, hrq1Dsgs1D, hrq1Dsgs1-K706A, hrq1-K318Asgs1D, and hrq1-K318Asgs1-K706A) to the S. cerevisiae single gene deletion and temperature-sensitive allele collections to generate double and triple mutants and scored them for synthetic positive and negative genetic effects based on colony growth. These screens identified hundreds of synthetic interactions, supporting the known roles of Hrq1 and Sgs1 in DNA repair, as well as suggesting novel connections to rRNA processing, mitochondrial DNA maintenance, transcription, and lagging strand synthesis during DNA replication.

DNA replication initiation factor RECQ4 possesses a role in antagonizing DNA replication initiation

“…If so, it would be important to determine how cells switch between RECQ4-APC/C and RECQ4-EMI1 complexes during these processes, and if this regulation involves post-translational modifications. It is worth noting that APC2, a component of the APC/C complex, has been detected in a synthetic genetic array analysis of the yeast Hrq1 (RECQ4 homolog) protein complex 54 . It remains to be determined if the role of RECQ4-APC/C interaction in replication initiation is conserved in yeast.…”

Phenomics approaches to understand genetic networks and gene function in yeast

“…In addition to double mutant genetic interactions, fitness-based phenomics is being used to systematically explore more complex genetic scenarios. For example, the SGA method was recently adapted to survey triple mutant genetic interactions, providing insight into the prevalence of trigenic interactions, the mechanisms of retention of duplicated genes, and the functional relationships between distant biological processes [36][37][38][39][40]. In addition, measurements of GIs in a variety of stress and other environmental conditions revealed that the GI network is remarkably robust, with most conditional GIs being detectable in the reference network but exacerbated or suppressed in different growth conditions [41].…”