A Network of Multi-Tasking Proteins at the DNA Replication Fork Preserves Genome Stability

Abstract

To elucidate the network that maintains high fidelity genome replication, we have introduced two conditional mutant alleles of DNA2, an essential DNA replication gene, into each of the approximately 4,700 viable yeast deletion mutants and determined the fitness of the double mutants. Fifty-six DNA2-interacting genes were identified. Clustering analysis of genomic synthetic lethality profiles of each of 43 of the DNA2-interacting genes defines a network (consisting of 322 genes and 876 interactions) whose topology provides clues as to how replication proteins coordinate regulation and repair to protect genome integrity. The results also shed new light on the functions of the query gene DNA2, which, despite many years of study, remain controversial, especially its proposed role in Okazaki fragment processing and the nature of its in vivo substrates. Because of the multifunctional nature of virtually all proteins at the replication fork, the meaning of any single genetic interaction is inherently ambiguous. The multiplexing nature of the current studies, however, combined with follow-up supporting experiments, reveals most if not all of the unique pathways requiring Dna2p. These include not only Okazaki fragment processing and DNA repair but also chromatin dynamics. Maintenance of genome stability from generation to generation is a primary defense against mutation and ensuing disease. Thus, the cell has evolved complex mechanisms, consisting of redundant, partially overlapping pathways, to protect the fidelity of genome inheritance. Using modern genetic screening techniques that allow one to investigate every gene in yeast that might be involved in these pathways, the researchers have defined a network consisting of 322 genes that together safeguard the DNA replication process. Previous approaches were limited to defining the interaction of one or a few genes, but the availability of mutants affecting all of the nonessential yeast genes allowed the identification of over 800 interactions in this study. In addition, the synthetic genetic array technique used in this study allowed identification of every nonessential gene in yeast that interacts with an essential replication protein, Dna2p. The comprehensiveness of the approach identified most, if not all, of the pathways in which the multitasking Dna2p participates, in a single experiment. The genomic scale of the study significantly accelerates understanding of this protein over traditional, low-throughput genetic methods.