Premium
Phosphorylation of the Sae2 endonuclease promotes its solubility and activity in DNA repair (735.4)
Author(s) -
Paull Tanya,
Fu Qiong
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.735.4
Subject(s) - endonuclease , cyclin dependent kinase , dna , nuclease , phosphorylation , biology , dna damage , yeast , microbiology and biotechnology , mutagenesis , biochemistry , cell cycle , mutation , cell , gene
The Sae2 protein is an endonuclease that is important for DNA double‐strand break processing together with the Mre11/Rad50/Xrs2 (MRX) complex in budding yeast. Sae2 is phosphorylated by cyclin‐dependent kinase, and this modification was shown to be essential for the coordination of DNA end resection with the S and G2 phases of the cell cycle. Here we map all of the modifications on Sae2 isolated from yeast, finding that there are constitutive as well as damage‐induced phosphorylation and acetylation events. Mutagenesis of these sites indicates that a C‐terminal cluster is most essential for the function of Sae2 in DNA damage survival, and reconstitution of Tel1 (ATM) activity on Sae2 with CDK and MRX in vitro shows that CDK phosphorylation primes these Tel1 modifications. From our experience with recombinant Sae2 expressed in E. coli, we knew that Sae2 forms a variety of oligomeric forms and that the smaller forms are more active than the larger forms in DNA binding and endonuclease activity. Here we find that Sae2 also forms oligomeric complexes in yeast, that these convert transiently into smaller units during DNA damage, and that mutation of the CDK and Tel1 phosphorylation sites alters this transition. Similarly we find that a large percentage of Sae2 appears to be insoluble in the absence of DNA damage, but a pool of Sae2 is transiently soluble. From these results we develop a working model of Sae2 regulation in which a potentially toxic, structure‐specific nuclease is held in an inactive state in normal cells but can be quickly released by post‐translational modifications to become active. Rapid degradation (through both the proteasome and through autophagy) then removes the active protein, returning the pool to an inactive state.