Search

Obstructions to replication fork progression, referred to collectively as DNA replication stress, challenge genome stability. In Saccharomyces cerevisiae, cells lacking RTT107 or SLX4 show genome instability and sensitivity to DNA replication stress and are defective in the completion of DNA replication during recovery from replication stress. We demonstrate that Slx4 is recruited to chromatin behind stressed replication forks, in a region that is spatially distinct from that occupied by the replication machinery. Slx4 complex formation is nucleated by Mec1 phosphorylation of histone H2A, which is recognized by the constitutive Slx4 binding partner Rtt107. Slx4 is essential for recruiting the Mec1 activator Dpb11 behind stressed replication forks, and Slx4 complexes are important for full activity of Mec1. We propose that Slx4 complexes promote robust checkpoint signaling by Mec1 by stably recruiting Dpb11 within a discrete domain behind the replication fork, during DNA replication stress., (© 2015 The Authors.)

DNA damage response pathways are crucial for protecting genome stability in all eukaryotes. Saccharomyces cerevisiae Dna2 has both helicase and nuclease activities that are essential for Okazaki fragment maturation, and Dna2 is involved in long-range DNA end resection at double-strand breaks. Dna2 forms nuclear foci in response to DNA replication stress and to double-strand breaks. We find that Dna2-GFP focus formation occurs mainly during S phase in unperturbed cells. Dna2 colocalizes in nuclear foci with 25 DNA repair proteins that define recombination repair centers in response to phleomycin-induced DNA damage. To systematically identify genes that affect Dna2 focus formation, we crossed Dna2-GFP into 4293 nonessential gene deletion mutants and assessed Dna2-GFP nuclear focus formation after phleomycin treatment. We identified 37 gene deletions that affect Dna2-GFP focus formation, 12 with fewer foci and 25 with increased foci. Together these data comprise a useful resource for understanding Dna2 regulation in response to DNA damage., (Copyright © 2015 Yimit et al.)