The replication stress and DNA damage checkpoint comprises crucial cellular responses for the maintenance of genome stability and prevention of carcinogenesis. Cellular challenges like stalled DNA polymerases and deficient DNA repair can result in persistent regions of ssDNA bound by RPA, which then recruits the essential kinase ATR. ATR plays a key role in the cell cycle checkpoint, DNA damage responses, replication fork stability, and control of origin firing. For the checkpoint to be triggered in S. cerevisiae , Mec1 (ATR homolog) must first encounter at least one of the three Mec1-activating proteins: Ddc1, Dpb11, and Dna2. Previous work in the field has demonstrated the important roles of these proteins in the damage response, but many questions remain about the dynamics and mechanics of how these early signaling factors find each other and assemble on damaged DNA. To address this, we have used a combination of single-molecule confocal microscopy and optical tweezers to follow the recruitment of fluorescent Mec1, 9-1-1, and Dpb11 to DNA with a 3 kb single-stranded gap in real time. Our findings support a model in which these proteins are primarily recruited to RPA-coated ssDNA via a three-dimensional search. We have characterized novel interactions between Dpb11 and gapped DNA. Furthermore, we observe that Dpb11 recruits Mec1 to gap junctions. These results have important implications for the ability of Mec1 to colocalize with its activators on DNA as required for checkpoint functioning and protection against DNA damage and replication stress.
Beckwitt et al. (Sun,) studied this question.