The Ddc1 checkpoint clamp subunit activates Mec1 kinase during the DNA damage checkpoint

Abstract

The DNA damage checkpoint pathway operates to prevent cell cycle progression in response to various genotoxic agents. In Saccharomyces cerevisiae, Mec1‐Ddc2 (human ATR‐ATRIP) is the kinase that initiates most checkpoint pathways. The checkpoint clamp is a heterotrimeric ring, Rad17‐Mec3‐Ddc1 (human 9‐1‐1), and shows structural similarity to the replication clamp PCNA. It is recruited to gapped DNA by the Rad24‐RFC clamp loader and is required for activation of the effector kinase Rad53 (human Chk1 or Chk2) in response to DNA damage.In vitro, the checkpoint clamp is loaded in an ATP‐dependent step onto 5’‐junctions of partial duplex DNA by the Rad24‐RFC clamp loader. The loaded clamp serves to activate Mec1 kinase activity. Activated Mec1 phosphorylates the Ddc1 and Mec3 subunits of the clamp, the Rad24 subunit of the loader, the Rpa1 and Rpa2 subunits of RPA, and the downstream kinase Rad53. We have used this activation system to probe the nature and functionality of interactions between Ddc1 and Mec1.