EXO1 is critical for embryogenesis and the DNA damage response in mice with a hypomorphic Nbs1 allele.

The maintenance of genome stability is critical for the suppression of diverse human pathologies that include developmental disorders, premature aging, infertility and predisposition to cancer. The DNA damage response (DDR) orchestrates the appropriate cellular responses following the detection of lesions to prevent genomic instability. The MRE11 complex is a sensor of DNA double strand breaks (DSBs) and plays key roles in multiple aspects of the DDR, including DNA end resection that is critical for signaling and DNA repair. The MRE11 complex has been shown to function both upstream and in concert with the 5'-3' exonuclease EXO1 in DNA resection, but it remains unclear to what extent EXO1 influences DSB responses independently of the MRE11 complex. Here we examine the genetic relationship of the MRE11 complex and EXO1 during mammalian development and in response to DNA damage. Deletion of Exo1 in mice expressing a hypomorphic allele of Nbs1 leads to severe developmental impairment, embryonic death and chromosomal instability. While EXO1 plays a minimal role in normal cells, its loss strongly influences DNA replication, DNA repair, checkpoint signaling and damage sensitivity in NBS1 hypomorphic cells. Collectively, our results establish a key role for EXO1 in modulating the severity of hypomorphic MRE11 complex mutations.

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

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.

EXO1 suppresses double-strand break induced homologous recombination between diverged sequences in mammalian cells.

DNA double-strand breaks (DSBs) can be repaired through several mechanisms, including homologous recombination (HR). While HR between identical sequences is robust in mammalian cells, HR between diverged sequences is suppressed by DNA mismatch-repair (MMR) components such as MSH2. Exonuclease I (EXO1) interacts with the MMR machinery and has been proposed to act downstream of the mismatch recognition proteins in mismatch correction. EXO1 has also been shown to participate in extensive DSB end resection, an initial step in the HR pathway. To assess the contribution of EXO1 to HR in mammalian cells, DSB-inducible reporters were introduced into Exo1-/- mouse embryonic stem cells, including a novel GFP reporter containing several silent polymorphisms to monitor HR between diverged sequences. Compared to HR between identical sequences which was not clearly affected, HR between diverged sequences was substantially increased in Exo1-/- cells although to a lesser extent than seen in Msh2-/- cells. Thus, like canonical MMR proteins, EXO1 can restrain aberrant HR events between diverged sequence elements in the genome.

Mouse models for human DNA mismatch-repair gene defects.

The mammalian DNA mismatch-repair genes belong to a family of genes that comprise several homologs of the Escherichia coli mutS and mutL genes. The observation that mutations in the two human repair genes MSH2 and MLH1 are responsible for hereditary nonpolyposis colorectal cancer, as well as a significant number of sporadic colorectal cancers, raises several questions about the role of these proteins and their family members in the initiation and progression of colorectal cancer. To address these questions, mice with inactivating mutations in all the known mutS and mutL homologs have been generated. The development of these mouse lines has permitted the systematic analysis of the role of each gene in the repair process and has underscored their significance in mutation avoidance and cancer susceptibility. These analyses were critical for our understanding of the function of these genes at the organismal level and also revealed an essential role for some of the DNA mismatch-repair genes in mammalian meiosis.

Maturational arrest of thymocyte development is caused by a deletion in the receptor-like protein tyrosine phosphatase kappa gene in LEC rats.

The Long-Evans Cinnamon (LEC) rat has a spontaneous mutation, T helper immunodeficiency (thid), which causes a markedly reduced CD4(+) thymocyte population. Here we positionally clone the locus and identify a deletion in the gene encoding a receptor-like protein tyrosine phosphatase kappa (Ptprk) that led to complete loss of the transcript. The rat Ptprk gene exhibits 98% identity with the human and mouse counterparts and is expressed most abundantly in the CD4(-)CD8(-) double-negative stage. The downregulation of Ptprk in mouse immature thymocytes by RNA interference mimicked the thid phenotype. These results indicate that thid maps to the Ptprk locus and that functional Ptprk is crucial for lineage commitment or progression of CD4(+) T cells. We also found that Ptprk appears to function in parallel with or downstream of Th-POK/cKrox (also known as ZBTB7B), a master regulator of T cell lineage decision.

Exonuclease-1 deletion impairs DNA damage signaling and prolongs lifespan of telomere-dysfunctional mice.

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.

Altered somatic hypermutation and reduced class-switch recombination in exonuclease 1-mutant mice.

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.

Inactivation of Exonuclease 1 in mice results in DNA mismatch repair defects, increased cancer susceptibility, and male and female sterility.

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.