Hydrolytic function of Exo1 in mammalian mismatch repair

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.

Single-molecule imaging reveals the mechanism of Exo1 regulation by single-stranded DNA binding proteins

“…Fifty-six percent of all hExo1 molecules (n = 244/435) localized to the vicinity of the 3′-ssDNA ends (Fig. 1C); the remaining nucleases were distributed at internal binding sites, consistent with hExo1's role in binding DNA nicks during MMR and NER (7,38). These data indicate that individual hExo1 molecules preferentially bind 3′-ssDNA overhangs but can also bind rare DNA nicks (we estimate approximately three to five per DNA molecule) that occur as a result of handling the 48-kb-long λ-DNA substrates.…”

“…Exo1, a member of the Rad2 family of nucleases, participates in DNA mismatch repair (MMR), double-strand break (DSB) repair, nucleotide excision repair (NER), and telomere maintenance (1)(2)(3). Exo1 is the only nuclease implicated in MMR, where its 5ʹ to 3ʹ exonuclease activity is used to remove long tracts of mismatch-containing single-stranded DNA (ssDNA) (2,(4)(5)(6)(7). In addition, functionally deficient Exo1 variants have been identified in familial colorectal cancers, and Exo1-null mice exhibit a significant increase in tumor development, decreased lifespan, and sterility (8,9).…”

MCM9 Is Required for Mammalian DNA Mismatch Repair

“…In the situation where the mismatch-containing strand possesses nicks on both sides, MCM9 helicase activity could in principle be sufficient to excise the mismatch strand (Song et al, 2010). However, the degradation of the mismatch-containing strand through the hydrolytic activity of Exo1 is known to be important for excision and MMR (Bregenhorn and Jiricny, 2014;Schaetzlein et al, 2013;Shao et al, 2014). The enzymatic action of MCM9 is expected to yield a 5 0 flapped single-stranded DNA that would be an optimal substrate on which Exo1 could act (Song et al, 2010).…”

DNA mismatch repair and the DNA damage response

“…Although an EXO1-MSH2/MLH1 interaction at physiological protein levels is not detected, ectopic expression of a nuclease-dead EXO1 [JL6] construct in MEF's in which endogenous EXO1 is absent restores a MSH2-CHK1 interaction and MNNG sensitivity [87]. A homozygous knock-in mouse harboring the Exo1-E109IK mutation is MMR-proficient but corresponding embryonic fibroblasts resemble those from Exo1 null strains with regard to loss of apoptotic signaling in response to various DNA damaging agents [74,84] The EXO1 E1091K mutant protein has subsequently been shown to be catalytically competent for excision suggesting that the loss of the DDR could be attributable to an as yet undefined structural role for Exo1 in this pathway [88,89]. Thus, a role for the scaffolding function of EXO1 in direct signaling is not ruled out.…”