Biochemical characterization of a cancer-associated E109K missense variant of human exonuclease 1.
Bottom Line: Because not all LS families carry mutations in these four genes, the search for cancer-associated mutations was extended to genes encoding other members of the mismatch repairosome.We now report that, contrary to earlier reports, and unlike the catalytic site mutant D173A, the EXO1 E109K variant resembled the wild-type (wt) enzyme on all tested substrates.In the light of our findings, we attempt here to reinterpret the results of the phenotypic characterization of a knock-in mouse carrying the E109K mutation and cells derived from it.
Affiliation: Institute of Molecular Cancer Research of the University of Zurich and the ETH Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.Show MeSH
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Mentions: Exonuclease 1 belongs to the RAD2 family of structure-specific endonucleases (33), the N-terminal and internal nuclease domains of which are highly conserved from bacteriophage to man (33,34). EXO1 possesses 5′→3′ exonuclease and flap endonuclease activities (35) that share the same active site, composed of two Mg2+ atoms coordinated by five aspartate residues. In human EXO1, these are D30, D152, D171, D173 and D225 (36). Glutamate 109 is not part of the active site; it resides in a flexible loop between α-helices α4 and α5 (Figure 1A) that form, together with the β-sheet β3, a mobile microdomain, which was postulated to mediate protein/protein interactions (36). As the E109K mutation replaces a negatively charged residue with a positively charged one, we wanted to test whether it perturbs intermolecular protein/protein interactions between EXO1 and its partners during MMR.
Affiliation: Institute of Molecular Cancer Research of the University of Zurich and the ETH Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.