Genetic Interaction Landscape Reveals Critical Requirements for Schizosaccharomyces pombe Brc1 in DNA Damage Response Mutants

• Published in: G3 : genes - genomes - genetics 2015-03, Vol.5 (5), p.953-962
• Main Authors: Sánchez, Arancha, Roguev, Assen, Krogan, Nevan J, Russell, Paul
• Format: Article
• Language: English
• Subjects: DNA Damage; Epistasis, Genetic - drug effects; Gene Expression; Gene Knockout Techniques; Gene Ontology; Histones - metabolism; Investigations; Mutagens - pharmacology; Mutation; Rad52 DNA Repair and Recombination Protein - genetics; Rad52 DNA Repair and Recombination Protein - metabolism; Replication Protein A - genetics; Replication Protein A - metabolism; Schizosaccharomyces - genetics; Schizosaccharomyces - metabolism; Schizosaccharomyces pombe Proteins - genetics; Schizosaccharomyces pombe Proteins - metabolism
• ISSN: 2160-1836; 2160-1836
• DOI: 10.1534/g3.115.017251

Abstract Brc1, which was first identified as a high-copy, allele-specific suppressor of a mutation impairing the Smc5-Smc6 holocomplex in Schizosaccharomyces pombe, protects genome integrity during normal DNA replication and when cells are exposed to toxic compounds that stall or collapse replication forks. The C-terminal tandem BRCT (BRCA1 C-terminus) domain of fission yeast Brc1 docks with phosphorylated histone H2A (γH2A)-marked chromatin formed by ATR/Rad3 checkpoint kinase at arrested and damaged replication forks; however, how Brc1 functions in relation to other genome protection modules remains unclear. Here, an epistatic mini-array profile reveals critical requirements for Brc1 in mutants that are defective in multiple DNA damage response pathways, including checkpoint signaling by Rad3-Rad26/ATR-ATRIP kinase, DNA repair by Smc5-Smc6 holocomplex, replication fork stabilization by Mrc1/claspin and Swi1-Swi3/Timeless-Tipin, and control of ubiquitin-regulated proteolysis by the COP9 signalosome (CSN). Exogenous genotoxins enhance these negative genetic interactions. Rad52 and RPA foci are increased in CSN-defective cells, and loss of γH2A increases genotoxin sensitivity, indicating a critical role for the γH2A-Brc1 module in stabilizing replication forks in CSN-defective cells. A negative genetic interaction with the Nse6 subunit of Smc5-Smc6 holocomplex indicates that the DNA repair functions of Brc1 and Smc5-Smc6 holocomplex are at least partially independent. Rtt107, the Brc1 homolog in Saccharomyces cerevisiae, has a very different pattern of genetic interactions, indicating evolutionary divergence of functions and DNA damage responses.