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

ABSTRACT 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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    • "These extract studies did not address the issue of structural versus catalytic involvement of the enzyme in mammalian repair, but analysis of several reconstituted human mismatch repair systems has demonstrated that the hydrolytic function of Exo1 plays an important role in the excision step of the reaction (9,11,13). The recent report that involvement of mouse Exo1 in mismatch repair is restricted to a structural role (28) was therefore surprising. "
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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.
    Nucleic Acids Research 05/2014; 42(11). DOI:10.1093/nar/gku420 · 9.11 Impact Factor
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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.
    Nucleic Acids Research 05/2014; 42(11). DOI:10.1093/nar/gku419 · 9.11 Impact Factor
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    • "Therefore, Exo1 has been considered to be the first exonuclease to be involved in SHM and CSR (Bardwell et al., 2004). Recently research showed that structural function and nuclease activity of mammalian Exo1 play differential roles in distinct DNA repair processes and tumor genesis in vivo (Schaetzlein et al., 2013). However, the effects of Exo1 on tissue stem cell homeostasis are largely unknown. "
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    ABSTRACT: Exonuclease 1 (Exo1) has been implicated in the regulation of DNA damage responses in stem cells with dysfunctional telomeres. However, it is unclear whether Exo1-mediated DNA maintenance pathways play a role in the maintenance of genomic stability and the self-renewal of tissue stem cells in mice with functional telomeres. Here, we analyzed the proliferative capacity of neural stem cells (NSCs) and hematopoietic stem cells (HSCs) from Exo1(-/-) mice. Our study shows that Exo1 deficiency impairs the maintenance of genomic stability and proliferative capacity in NSCs but not HSCs. In line with these results, we detected a decrease in proliferation and an up-regulation of p21 expression levels in Exo1-deficient NSCs but not Exo1-deficient HSCs. Our data provide experimental evidence that Exo1 deficiency has a differential impact on the homeostasis and proliferative capacity of tissue stem cells in the brain and bone marrow, suggesting that different tissue stem cells utilize distinct mechanisms for maintaining their genomic stability. Our current study provides important insight into the role of Exo1-mediated DNA maintenance pathways in the maintenance of genomic stability and the proliferative capacity of tissue stem cells.
    Stem Cell Research 11/2013; 12(1):250-259. DOI:10.1016/j.scr.2013.11.001 · 3.69 Impact Factor
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