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.
Highlights
In order to preserve the fidelity of genome duplication during DNA replication, cells with complex genomes have evolved a network of pathways composed of the DNA replication apparatus, DNA repair proteins, and regulatory activities
Sister Chromatid Cohesion and Repair of double-strand break (DSB) in the rDNA Ctf4p is a pol a–binding protein [50], and ctf4D strains are defective in sister chromatid cohesion [20]. dna2–2 was identified as a mutant synthetically lethal with ctf4D, but we have shown that dna2 mutants are not defective in cohesion [51]
We previously reported that the dna2-2 mutation gave rise to an increased frequency of DSBs at the replication fork barrier (RFB) in the rDNA and that deleting FOB1, which is required for pausing at the RFB, suppressed DSB formation
Summary
In order to preserve the fidelity of genome duplication during DNA replication, cells with complex genomes have evolved a network of pathways composed of the DNA replication apparatus, DNA repair proteins, and regulatory activities. While the most familiar example is the proofreading activity found in the DNA polymerases, other proteins of the replisome have evolved substrate specificities to address errors made during replication fork progression. One of these proteins is Dna2p, a helicase/nuclease. It has been proposed that Dna2p acts with FEN1 to remove RNA primers from Okazaki fragments whose 59 RNA/DNA termini have been extensively displaced by DNA polymerase (pol) d. Dna2p co-purifies with FEN1, which is a structure-specific nuclease required for OFP in the SV40 in vitro replication system [5,6].
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