Abstract
The eukaryotic DNA damage and replication stress checkpoint is an essential component of the DNA damage response and crucial for genome maintenance. In budding yeast, the apical kinase Mec1 (ATR ortholog), along with binding partner Ddc2 (ATRIP ortholog), senses persistent RPA-bound ssDNA in the cell. Mec1 is activated by interaction with a Mec1-activating protein. One such activator, Dpb11 (TopBP1 ortholog), is recruited to a 5' ss-dsDNA junction via the 9-1-1 checkpoint clamp. Due to their differential DNA binding preferences, it remains to be determined how Mec1 encounters its activators on damaged DNA. Using real-time single-molecule imaging of checkpoint proteins binding to dsDNA containing a long ssDNA gap, we show that, even in the absence of 9-1-1, Dpb11 binds to ssDNA and localizes to ss-dsDNA junctions in an RPA-dependent manner. Importantly, we directly visualize that Dpb11 recruits Mec1-Ddc2 to ss-dsDNA junctions. Additionally, single-molecule force spectroscopy was used to demonstrate that Dpb11 can interact with multiple DNA sites simultaneously to form bridges both alone and in the presence of RPA, stabilizing ssDNA loops and reducing the end-to-end distance of gapped DNA. Taken together, these data support a model in which Dpb11 facilitates Mec1 colocalization with its activators both directly by recruiting Mec1 to gap junctions and indirectly by decreasing the effective gap length.