Mammalian Exo1 encodes both structural and catalytic functions that play distinct roles in essential biological processes

Departments of Cell Biology and Pathology, Albert Einstein College of Medicine, Bronx, NY 10461.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 06/2013; 110(27). DOI: 10.1073/pnas.1308512110
Source: PubMed


Mammalian Exonuclease 1 (EXO1) is an evolutionarily conserved, multifunctional exonuclease involved in DNA damage repair, replication, immunoglobulin diversity, meiosis, and telomere maintenance. It has been assumed that EXO1 participates in these processes primarily through its exonuclease activity, but recent studies also suggest that EXO1 has a structural function in the assembly of higher-order protein complexes. To dissect the enzymatic and nonenzymatic roles of EXO1 in the different biological processes in vivo, we generated an EXO1-E109K knockin (Exo1(EK)) mouse expressing a stable exonuclease-deficient protein and, for comparison, a fully EXO1-deficient (Exo1(null)) mouse. In contrast to Exo1(null/null) mice, Exo1(EK/EK) mice retained mismatch repair activity and displayed normal class switch recombination and meiosis. However, both Exo1-mutant lines showed defects in DNA damage response including DNA double-strand break repair (DSBR) through DNA end resection, chromosomal stability, and tumor suppression, indicating that the enzymatic function is required for those processes. On a transformation-related protein 53 (Trp53)-null background, the DSBR defect caused by the E109K mutation altered the tumor spectrum but did not affect the overall survival as compared with p53-Exo1(null) mice, whose defects in both DSBR and mismatch repair also compromised survival. The separation of these functions demonstrates the differential requirement for the structural function and nuclease activity of mammalian EXO1 in distinct DNA repair processes and tumorigenesis in vivo.

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Available from: R. Chahwan, Feb 25, 2015
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    • "Another difference is that although shortrange resection by the MRX is sufficient for efficient HR in S. cerevisiae (Mimitou & Symington 2008; Zhu et al. 2008), long-range resection by Dna2 seems to play an essential role in HR in mammalian and chicken cell lines (Peng et al. 2012; Hoa et al. 2015). Exo1 might play a minor role, as evidenced by the fact that Exo1-deficient mice develop normally (Schaetzlein et al. 2013). The relative contributions of vertebrate Mre11, CtIP, Dna2 and Exo1 to DSB resection have not yet been defined in any mammalian cell lines, as previous studies have relied on the incomplete depletion of the relevant proteins by siRNA. "
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    ABSTRACT: Homologous recombination (HR) is initiated by double-strand break (DSB) resection, during which DSBs are processed by nucleases to generate 3' single-strand DNA. DSB resection is initiated by CtIP and Mre11 followed by long-range resection by Dna2 and Exo1 in Saccharomyces cerevisiae. To analyze the relative contribution of four nucleases, CtIP, Mre11, Dna2 and Exo1, to DSB resection, we disrupted genes encoding these nucleases in chicken DT40 cells. CtIP and Dna2 are required for DSB resection, whereas Exo1 is dispensable even in the absence of Dna2, which observation agrees with no developmental defect in Exo1-deficient mice. Despite the critical role of Mre11 in DSB resection in S. cerevisiae, loss of Mre11 only modestly impairs DSB resection in DT40 cells. To further test the role of CtIP and Mre11 in other species, we conditionally disrupted CtIP and MRE11 genes in the human TK6 B cell line. As with DT40 cells, CtIP contributes to DSB resection considerably more significantly than Mre11 in TK6 cells. Considering the critical role of Mre11 in HR, this study suggests that Mre11 is involved in a mechanism other than DSB resection. In summary, CtIP and Dna2 are sufficient for DSB resection to ensure efficient DSB repair by HR.
    Full-text · Article · Nov 2015 · Genes to Cells
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    • "Although coupled action of the mismatch repair and damage signaling pathways has been implicated in the checkpoint and apoptotic responses to DNA methylator damage (44), the latter phenotype is indicative of a defect that is presumably distinct from conventional mismatch processing. Because Exo1-E109K was presumed to be catalytically defective, these phenotypes have been interpreted in terms of a requirement for Exo1 hydrolytic function in double-strand break repair and methylator damage response pathways (28). In view of our finding that Exo1-E109K is catalytically functional, we suggest that these phenotypes are due to structural consequences of the E109K mutation, which could for example directly or indirectly perturb Exo1 physical interaction with other components of these pathways. "
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    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.
    Full-text · Article · May 2014 · Nucleic Acids Research
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    • "EXO1 was also described to interact with CtIP, a polypeptide involved in the resection of DNA double-strand breaks (DSBs) (37). As mouse embryonic fibroblasts expressing the E109K mutant were reported to be hypersensitive to camptothecin (28), which induces DSBs during replication, we wanted to test whether the interaction of this variant with CtIP might be perturbed. However, as shown by far-western blotting (Figure 2B), all three EXO1 variants behaved similarly in this assay. "
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    ABSTRACT: Mutations in the mismatch repair (MMR) genes MSH2, MSH6, MLH1 and PMS2 are associated with Lynch Syndrome (LS), a familial predisposition to early-onset cancer of the colon and other organs. 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. This effort identified mutations in EXO1, which encodes the sole exonuclease implicated in MMR. One of these mutations, E109K, was reported to abrogate the catalytic activity of the enzyme, yet, in the crystal structure of the EXO1/DNA complex, this glutamate is far away from both DNA and the catalytic site of the enzyme. In an attempt to elucidate the reason underlying the putative loss of function of this variant, we expressed it in Escherichia coli, and tested its activity in a series of biochemical assays. 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.
    Full-text · Article · May 2014 · Nucleic Acids Research
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