Abstract
Genetic and biochemical studies have previously implicated exonuclease 1 (Exo1) in yeast and mammalian mismatch repair, with results suggesting that function of the protein in the reaction depends on both its hydrolytic activity and its ability to interact with other components of the repair system. However, recent analysis of an Exo1-E109K knockin mouse has concluded that Exo1 function in mammalian mismatch repair is restricted to a structural role, a conclusion based on a prior report that N-terminal His-tagged Exo1-E109K is hydrolytically defective. Because Glu-109 is distant from the nuclease hydrolytic center, we have compared the activity of untagged full-length Exo1-E109K with that of wild type Exo1 and the hydrolytically defective active site mutant Exo1-D173A. We show that the activity of Exo1-E109K is comparable to that of wild type enzyme in a conventional exonuclease assay and that in contrast to a D173A active site mutant, Exo1-E109K is fully functional in mismatch-provoked excision and repair. We conclude that the catalytic function of Exo1 is required for its participation in mismatch repair. We also consider the other phenotypes of the Exo1-E109K mouse in the context of Exo1 hydrolytic function.
Highlights
Exonuclease 1 (Exo1), a 5 to 3 hydrolytic activity of the Rad[2] family, has been implicated in multiple genetic stabilization pathways including mismatch repair, doublestrand break repair and telomere maintenance (reviewed in (1))
Whole cell extracts were prepared from immortalized Mlh1−/− Exo1−/− mouse embryo fibroblast (MEF) cells according to Kadyrov et al (18)
The Exo1-E109K mutation was identified in several putative hereditary non-polyposis colorectal cancer (HNPCC) families (20)
Summary
Exonuclease 1 (Exo1), a 5 to 3 hydrolytic activity of the Rad[2] family, has been implicated in multiple genetic stabilization pathways including mismatch repair, doublestrand break repair and telomere maintenance (reviewed in (1)). Analysis of extract reactions and biochemical reconstitution experiments have directly implicated Exo[1] in mammalian mismatch repair, with the latter experiments indicating involvement of Exo[1] hydrolysis during the excision step of repair (9–13). The simplest, in which excision is directed by a strand break 5 to the mispair, depends on MutS␣, MutL␣, Exo[1] and RPA (replication protein A) (11– 13). In this reaction, MutS␣ activates Exo[1] and renders the exonuclease highly processive (11,12). MutL␣ is not required for excision in this system but acts together with MutS␣ to modestly suppress hydrolysis on mismatch-free DNA, and the mismatch-dependence of excision can be further enhanced by the presence of PARP-1 (poly[ADPribose] polymerase 1) (14)
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