Skip to Content
Merck
  • Sae2 and Rif2 regulate MRX endonuclease activity at DNA double-strand breaks in opposite manners.

Sae2 and Rif2 regulate MRX endonuclease activity at DNA double-strand breaks in opposite manners.

Cell reports (2021-04-01)
Antonio Marsella, Elisa Gobbini, Corinne Cassani, Renata Tisi, Elda Cannavo, Giordano Reginato, Petr Cejka, Maria Pia Longhese
ABSTRACT

The Mre11-Rad50-Xrs2 (MRX) complex detects and processes DNA double-strand breaks (DSBs). Its DNA binding and processing activities are regulated by transitions between an ATP-bound state and a post-hydrolysis cutting state that is nucleolytically active. Mre11 endonuclease activity is stimulated by Sae2, whose lack increases MRX persistence at DSBs and checkpoint activation. Here we show that the Rif2 protein inhibits Mre11 endonuclease activity and is responsible for the increased MRX retention at DSBs in sae2Δ cells. We identify a Rad50 residue that is important for Rad50-Rif2 interaction and Rif2 inhibition of Mre11 nuclease. This residue is located near a Rad50 surface that binds Sae2 and is important in stabilizing the Mre11-Rad50 (MR) interaction in the cutting state. We propose that Sae2 stimulates Mre11 endonuclease activity by stabilizing a post-hydrolysis MR conformation that is competent for DNA cleavage, whereas Rif2 antagonizes this Sae2 function and stabilizes an endonuclease inactive MR conformation.

MATERIALS
Product Number
Brand
Product Description

Millipore
D-(+)-Raffinose pentahydrate, suitable for microbiology, ≥99.0%, Microbiological media component
Sigma-Aldrich
Sodium deoxycholate, BioXtra, ≥98.0% (dry matter, NT)
Sigma-Aldrich
Phospho(enol)pyruvic acid trisodium salt hydrate, ≥97% (enzymatic)
Roche
Hygromycin B, from Streptomyces hygroscopicus
Sigma-Aldrich
DL-Dithiothreitol, ≥99.0% (RT)
Millipore
D-(+)-Glucose monohydrate, suitable for microbiology, ≥99.0%
Sigma-Aldrich
Phenylmethanesulfonyl fluoride, ≥99.0% (T)
Sigma-Aldrich
D-(+)-Maltose monohydrate, ≥99% (HPLC), BioXtra
Sigma-Aldrich
Protease Inhibitor Cocktail, for use with mammalian cell and tissue extracts, DMSO solution
Roche
cOmplete, Mini Protease Inhibitor Cocktail, Tablets provided in a glass vial