Hydrolytic function of Exo1 in mammalian mismatch repair.
Bottom Line: 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 also consider the other phenotypes of the Exo1-E109K mouse in the context of Exo1 hydrolytic function.
Affiliation: Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.Show MeSH
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Mentions: Supplementation of MutSα, MutLα, Exo1, RPA, PCNA and RFC, the components required for bidirectional excision, with DNA polymerase δ and the dNTPs yields a minimal system that supports both 5′- and 3′-directed mismatch repair (37). As shown in Figure 5, wild type Exo1 and Exo1-E109K support MutSα-dependent mismatch correction on 5′- (panel A, compare lanes 4 and 5 with 8 and 9) or 3′-heteroduplex DNA (panel B, compare lanes 6 and 7, and lanes 8 and 9). However, as observed in cell extracts, hydrolytically deficient Exo1-D173A is incapable of supporting the repair of either heteroduplex in this purified system (panel A, lane 3; panel B, lane 4).
Affiliation: Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.