Slx4 Regulates DNA Damage Checkpoint-dependent Phosphorylation of the BRCT Domain Protein Rtt107/Esc4

Abstract

RTT107 (ESC4, YHR154W) encodes a BRCA1 C-terminal-domain protein that is important for recovery from DNA damage during S phase. Rtt107 is a substrate of the checkpoint protein kinase Mec1, although the mechanism by which Rtt107 is targeted by Mec1 after checkpoint activation is currently unclear. Slx4, a component of the Slx1-Slx4 structure-specific nuclease, formed a complex with Rtt107. Deletion of SLX4 conferred many of the same DNA-repair defects observed in rtt107Δ, including DNA damage sensitivity, prolonged DNA damage checkpoint activation, and increased spontaneous DNA damage. These phenotypes were not shared by the Slx4 binding partner Slx1, suggesting that the functions of the Slx4 and Slx1 proteins in the DNA damage response were not identical. Of particular interest, Slx4, but not Slx1, was required for phosphorylation of Rtt107 by Mec1 in vivo, indicating that Slx4 was a mediator of DNA damage-dependent phosphorylation of the checkpoint effector Rtt107. We propose that Slx4 has roles in the DNA damage response that are distinct from the function of Slx1-Slx4 in maintaining rDNA structure and that Slx4-dependent phosphorylation of Rtt107 by Mec1 is critical for replication restart after alkylation damage.