MammalianExo1encodes both structural and catalytic functions that play distinct roles in essential biological processes

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.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...

Alternative End-Joining and Classical Nonhomologous End-Joining Pathways Repair Different Types of Double-Strand Breaks during Class-Switch Recombination

“…EXO1 requires a nick and would continue until it finds an SSB in the opposite strand (44,54), but recent evidence indicates that EXO1 functions as a scaffold in by guest on May 9, 2018 http://www.jimmunol.org/ CSR for an unknown exonucleases that would then generate a DSB with a single-strand end or overhang. Mammalian EXO1 encodes both structural and catalytic functions that play distinct roles in essential biological processes (55). In support of the proposed role for the MMR pathway during CSR, the increase in EXO1 and RPA recruitment in the S-regions observed in AID low cells occurs at later times and correlates with the decrease in the density of mutations in the S-regions found in these cells (Figs.…”

“…One potential explanation for this is that during CSR, CtIP is required to maintain the resected ends generated by the MMR pathway, as was recently suggested in another system (56). Alternatively, this could be explained by the scaffold role of EXO1 in CSR (55). Our results in AID low cells agree with previous evidence showing decreased microhomology in switch junctions from MSH2-and EXO1-deficient mice (57,58) as well as with more recent evidence that MMR functions upstream of the DSB in C-NHEJ mutant cells (45) (Fig.…”

Coupling of Human DNA Excision Repair and the DNA Damage Checkpoint in a Defined in Vitro System

“…The Exo1 Ϫ/Ϫ MEFs were obtained from Winfried Edelmann (Albert Einstein College of Medicine) (27). To drive cells into quiescence, cells were grown in DMEM containing 0.5% FBS for 3-4 days.…”

14-3-3 Proteins Restrain the Exo1 Nuclease to Prevent Overresection

“…Its function has been implicated in a wide range of biological processes, including genome maintenance, meiosis, and telomere regulation as well as class switch recombination and somatic hypermutation in lymphocytes (21,22). Although Exo1 deficiency causes defects in DNA repair and meiosis and an elevated susceptibility to cancer (23,24), inappropriately higher levels of Exo1 activity could also have detrimental effects. Consistent with this notion, deletion of Exo1 in yeast or mice reverses the phenotypes caused by loss of function of Cdc13 or telomerase (which results in uncapped or dysfunctional telomeres), including cell survival and life span (25)(26)(27).…”