Phosphorylation of Sae2 mediates FHA-specific interaction and regulates its DNA repair function [Genomics and Proteomics]

March 11th, 2015 by Liang, J., Suhandynata, R. T., Zhou, H.

Saccharomyces cerevisiae Sae2 and its ortholog CtIP in higher eukaryotes have a conserved role in the initial processing of DNA lesions and influencing their subsequent repair pathways. Sae2 is phosphorylated by the ATR/ATM family kinases Mec1 and Tel1 in response to DNA damage. Among the Mec1/Tel1 consensus phosphorylation sites of Sae2, we found that mutations of T90 and T279 of Sae2 into alanine caused a persistent Rad53 activation in response to a transient DNA damage, similar to the loss of Sae2. To gain insight into the function of this phosphorylation of Sae2, we performed a quantitative proteomics analysis to identify its associated proteins. We found that phosphorylation of T90 of Sae2 mediates its interaction with Rad53, Dun1, Xrs2, Dma1 and Dma2, whereas Rad53 and Dun1 additionally interact with phosphorylated T279 of Sae2. Mutations of the ligand-binding residues of FHA domains of Rad53, Dun1, Xrs2, Dma1 and Dma2 abolished their interactions with Sae2, revealing the involvement of FHA-specific interactions. Mutations of T90 and T279 of Sae2 caused a synergistic defect when combined with sgs1Δ and exo1Δ, and elevated gross chromosomal rearrangements (GCRs). Likewise, mutations of RAD53 and DUN1 caused a synthetic growth defect with sgs1Δ and elevated GCRs. These findings suggest that threonine-specific phosphorylation of Sae2 by Mec1 and Tel1 contributes to DNA repair and genome maintenance via its interactions with Rad53 and Dun1.