Assembly of Slx4 signaling complexes behind DNA replication forks

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. Synopsis The stepwise assembly of Slx4 protein complexes on chromatin during DNA replication stress promotes checkpoint signaling. Phosphorylation of histone H2A by the Mec1 checkpoint kinase nucleates the assembly of Rtt107‐Slx4‐Dpb11 complexes on chromatin during DNA replication stress. Rtt107‐Slx4‐Dpb11 complexes assemble behind stressed DNA replication forks at regions that are spatially distinct from the replisome. Slx4 recruits Dpb11 behind the fork to promote full activation of Mec1 kinase during DNA replication stress. Slx4 complexes stimulate the completion of DNA synthesis during replication stress. Graphical Abstract The stepwise assembly of Slx4 protein complexes on chromatin during DNA replication stress promotes checkpoint signaling.