Preparation of endogenous TopBP1/Dpb11 and effect on central checkpoint kinase Mec1- Ddc2 (human ATR-ATRIP homolog).

The Saccharomyces cerevisiae Mec1 kinase, the mammalian ATR ortholog, is essential for sensing a variety of DNA lesions and initiating DNA damage response. The Dpb11, a homolog of human TopBP1, functions in activating the Mec1 upon DNA replication stress and DNA damages. Here, we report an affinity purification and ion exchange chromatography method to efficiently purify endogenous Dpb11 under normal expression level directly from yeast whole cell extraction. The final concentration of 5 µM of high purity and homogeneity biochemical preparation enables functional and structural characterization of the physical interaction between Dpb11 and Mec1-Ddc2 complex. The Dpb11 obtained by endogenous purification strongly stimulates the Mec1 kinase activity and promotes the changes in conformational distribution. This observation suggests the Dpb11 activates Mec1 kinase probably through modulation in the kinase conformations.

3.9 Å structure of the yeast Mec1-Ddc2 complex, a homolog of human ATR-ATRIP.

The ataxia telangiectasia-mutated and Rad3-related (ATR) kinase is a master regulator of DNA damage response and replication stress in humans, but the mechanism of its activation remains unclear. ATR acts together with its partner ATRIP. Using cryo-electron microscopy, we determined the structure of intact Mec1-Ddc2 (the yeast homolog of ATR-ATRIP), which is poised for catalysis, at a resolution of 3.9 angstroms. Mec1-Ddc2 forms a dimer of heterodimers through the PRD and FAT domains of Mec1 and the coiled-coil domain of Ddc2. The PRD and Bridge domains in Mec1 constitute critical regulatory sites. The activation loop of Mec1 is inhibited by the PRD, revealing an allosteric mechanism of kinase activation. Our study clarifies the architecture of ATR-ATRIP and provides a structural framework for the understanding of ATR regulation.