Exonuclease 1 (Exo1) is a 5-3 exonuclease that interacts with MutS and MutL homologs and has been implicated in the excision step of DNA mismatch repair. To investigate the role of Exo1 in mammalian mismatch repair and assess its importance for tumorigenesis and meiosis, we generated an Exo1 mutant mouse line. Analysis of Exo1 −/− cells for mismatch repair activity in vitro showed that Exo1 is required for the repair of base:base and single-base insertion/deletion mismatches in both 5 and 3 nick-directed repair. The repair defect in Exo1 −/− cells also caused elevated microsatellite instability at a mononucleotide repeat marker and a significant increase in mutation rate at the Hprt locus. Exo1 −/− animals displayed reduced survival and increased susceptibility to the development of lymphomas. In addition, Exo1 −/− male and female mice were sterile because of a meiotic defect. Meiosis in Exo1 −/− animals proceeded through prophase I; however, the chromosomes exhibited dynamic loss of chiasmata during metaphase I, resulting in meiotic failure and apoptosis. Our results show that mammalian Exo1 functions in mutation avoidance and is essential for male and female meiosis. The DNA mismatch repair (MMR) system is important for maintaining the integrity of the genome in prokaryotes and eukaryotes. It has evolved to correct mispaired bases that result from errors during DNA replication, DNA recombination, and from certain types of DNA damage. Analyses in yeast and mice also revealed an essential role of some eukaryotic MMR gene products in the control of meiotic recombination. The importance of MMR to mammals is also highlighted by the observation that germ-line mutations in several of the MMR genes are associated with hereditary nonpolyposis colorectal cancer (HNPCC), and the loss of MMR function underlies several types of sporadic cancers (Peltomaki and Vasen 1997;Peltomaki 2001).MMR was initially characterized in bacteria (Modrich 1991;Modrich and Lahue 1996) and more recently in eukaryotic cells (Kolodner 1996). The studies of mammalian MMR focused mainly on the role of the eukaryotic MutS and MutL homologs (MSH and MLH, respectively) in the initiation of the repair reaction. These analyses showed that the early steps of MMR include the recognition of mispaired nucleotide(s) by two heterodimeric complexes: MSH2-MSH6 functions in the repair of base:base mispairs as well as a range of insertion/deletion loop mispairs (IDLs), whereas MSH2-MSH3 primarily functions in the repair of IDLs (Marsischky et al. 1996;Genschel et al. 1998;Umar et al. 1998). Subsequent to mismatch recognition, these two MutS complexes interact with MutL complexes consisting of MLH1-PMS2 or MLH1-MLH3 (Prolla et al. 1994;Li and Modrich 1995;Flores-Rozas and Kolodner 1998;Wang et al. 1999). These interactions are absolutely necessary for the activation of downstream events including the excision of the misincorporated nucleotide(s) and filling in of the resulting single-strand gap by DNA resynthesis. These later steps in MMR as well as the nature...

The generation of protective antibodies requires somatic hypermutation (SHM) and class-switch recombination (CSR) of immunoglobulin genes. Here we show that mice mutant for exonuclease 1 (Exo1), which participates in DNA mismatch repair (MMR), have decreased CSR and changes in the characteristics of SHM similar to those previously observed in mice mutant for the MMR protein Msh2. Exo1 is thus the first exonuclease shown to be involved in SHM and CSR. The phenotype of Exo1(-/-) mice and the finding that Exo1 and Mlh1 are physically associated with mutating variable regions support the idea that Exo1 and MMR participate directly in SHM and CSR.

Exonuclease-1 (EXO1) mediates checkpoint induction in response to telomere dysfunction in yeast, but it is unknown whether EXO1 has similar functions in mammalian cells. Here we show that deletion of the nuclease domain of Exo1 reduces accumulation of DNA damage and DNA damage signal induction in telomere-dysfunctional mice. Exo1 deletion improved organ maintenance and lifespan of telomere-dysfunctional mice but did not increase chromosomal instability or cancer formation. Deletion of Exo1 also ameliorated the induction of DNA damage checkpoints in response to gamma-irradiation and conferred cellular resistance to 6-thioguanine-induced DNA damage. Exo1 deletion impaired upstream induction of DNA damage responses by reducing ssDNA formation and the recruitment of Replication Protein A (RPA) and ATR at DNA breaks. Together, these studies provide evidence that EXO1 contributes to DNA damage signal induction in mammalian cells, and deletion of Exo1 can prolong survival in the context of telomere dysfunction.

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.somatic hypermuation | scaffold function | ssDNA E xonuclease 1 (EXO1) belongs to the XPG/Rad2 family of metallonucleases and was first described as a 5′-3′ exonuclease associated with meiosis in Schizosaccharomyces pombe (1). Since then EXO1 has been implicated in a multitude of eukaryotic DNA metabolic pathways and in maintaining genomic integrity. It is involved in DNA mismatch repair (MMR) by hydrolyzing DNA mismatches (2-4), in DNA double-strand break repair (DSBR) through DNA end resection (5-7), in B-cell development through the generation of antibody diversity (8), and in telomere maintenance by promotion of telomeric recombination (9). Biochemical analysis had shown that the N-terminal half of EXO1 possesses 5′-3′ exonuclease and 5′ flap-endonuclease activities (10). However, these apparently distinct functions now are thought to be mechanistically unified (11).MMR is essential for maintaining the integrity of eukaryotic genomes by removing misincorporated nucleotides that result from erroneous replication. During MMR, the repair of distinct types of mismatches is initiated by two partially redundant MutS homolog (MSH) complexes: the MSH2-MSH6 (MutSα) heterodimer, that recognizes and binds to single-base mispairs and single-base insertion/deletions, and the MSH2-MSH3 (MutSβ) complex that primarily interacts with single-base and larger insertions/deletions. Subsequent to mismatch recognition by the MSH complexes, a MutL homolog (MLH) complex con...

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