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 Lac repressor-operator interaction was used as a reversible DNA end-blocking system in conjunction with an IAsys biosensor instrument (Thermo Affinity Sensors), which detects total internal reflectance and allows monitoring of binding and dissociation in real time, in order to develop a system for studying the ability of mismatch repair proteins to move along the DNA. The MSH2-MSH6 complex bound to a mispaired base was found to be converted by ATP binding to a form that showed rapid sliding along the DNA and dissociation via the DNA ends and also showed slow, direct dissociation from the DNA. In contrast, the MSH2-MSH6 complex bound to a base pair containing DNA only showed direct dissociation from the DNA. The MLH1-PMS1 complex formed both mispair-dependent and mispair-independent ternary complexes with the MSH2-MSH6 complex on DNA. The mispair-independent ternary complexes were formed most efficiently on DNA molecules with free ends under conditions where ATP hydrolysis did not occur, and only exhibited direct dissociation from the DNA. The mispair-dependent ternary complexes were formed in the highest yield on DNA molecules with blocked ends, required ATP and magnesium for formation, and showed both dissociation via the DNA ends and direct dissociation from the DNA.Errors that occur during DNA replication result in base-base mismatches and small insertion/deletion mismatches that if left uncorrected are fixed in the DNA as mutations by subsequent rounds of DNA replication. The DNA mismatch repair (MMR) 1 system normally corrects such errors in the cell and is highly conserved from bacteria to humans (1-5). Defects in the system lead to increased rates of accumulation of mutations, and in humans, inherited and somatic defects in MMR result in increased development of cancer (6 -9). The mechanism of MMR is best understood in Escherichia coli (3)(4)(5). In E. coli the MutS protein, which appears to function as a homodimer, serves as the mispair recognition factor (10 -13). MutL, another homodimer, interacts with the MutS complex in the presence of a mispair and ATP and activates the endonucleolytic activity of MutH (11, 14 -20). MutH makes single strand breaks in the newly synthesized daughter strand at transiently unmethylated GATC sequences, allowing the unwinding of the DNA by UvrD coupled with the degradation of the error-containing strand by one of at least four redundant exonucleases (21, 22). Then DNA polymerase III holoenzyme can resynthesize the DNA strand leaving a nick that is subsequently sealed by DNA ligase (23). In eukaryotic cells, three different MutS homologs form two different heterodimeric complexes, called MSH2-MSH6 (MutS␣) and MSH2-MSH3 (MutS␤) that together recognize base-base mispairs and insertion/deletion loops (1, 2, 24 -35). Similarly, three different MutL homologs form two different heterodimeric complexes, called MLH1-PMS1 (MutL␣; PMS1 Saccharomyces cerevisiae is PMS2 in humans) and MLH1-MLH3, that function in MMR (36 -38). Like their bacterial homologs, the MSH and MLH complexe...

The 335 exonucleases catalyze the excision of nucleoside monophosphates from the 3 termini of DNA. We have identified the cDNA sequences encoding two 335 exonucleases (TREX1 and TREX2) from mammalian cells. The TREX1 and TREX2 proteins are 304 and 236 amino acids in length, respectively. Analysis of the TREX1 and TREX2 sequences identifies three conserved motifs that likely generate the exonuclease active site in these enzymes. The specific amino acids in these three conserved motifs suggest that these mammalian exonucleases are most closely related to the proofreading exonucleases of the bacterial replicative DNA polymerases and the RNase T enzymes. Expression of TREX1 and TREX2 in Escherichia coli demonstrates that these recombinant proteins are active 335 exonucleases. The recombinant TREX1 protein was purified, and exonuclease activity was measured using single-stranded, partial duplex, and mispaired oligonucleotide DNA substrates. The greatest activity of the TREX1 protein was detected using a partial duplex DNA containing five mispaired nucleotides at the 3 terminus. No activity was detected using single-stranded RNA or an RNA-DNA partial duplex. Identification of the TREX1 and TREX2 cDNA sequences provides the genetic tools to investigate the physiological roles of these exonucleases in mammalian DNA replication, repair, and recombination pathways.The multistep processes of DNA replication, repair, and recombination in human cells often require the excision of nucleotides from the DNA 3Ј termini. For each cell division 4 billion nucleotides must be correctly replicated. The polymerization of incorrect or structurally modified nucleotides into DNA generates the 3Ј termini that block chain elongation by the DNA polymerases. Oxidative damage to DNA can result in fragmented nucleotides at the 3Ј termini that can not be elongated by DNA polymerases. Genetic recombination and mismatch repair pathways can require the removal of normal nucleotides from the 3Ј termini of DNA chains. Enzymes containing 3Ј35Ј exonuclease activity remove these mismatched, modified, fragmented, and normal nucleotides to generate the appropriate 3Ј termini for subsequent steps in the DNA metabolic pathways.Several 3Ј35Ј exonucleases have been described from a variety of animal cells (1-5). These exonucleases demonstrate similar biochemical properties, but the relationships between these enzymes are not known. Also, there are 3Ј35Ј exonucleases contained in the structural domains of mammalian DNA pols 1 ␦ (6), ⑀ (7), and ␥ (8). These proofreading 3Ј35Ј exonucleases excise incorrectly polymerized nucleotides during DNA synthesis. Thus, a variety of 3Ј35Ј exonucleases are present in mammalian cells. These exonucleases might function in multiple pathways to generate 3Ј termini that support further steps such as polymerization or ligation.Excision of incorrectly polymerized nucleotides by proofreading 3Ј35Ј exonucleases is an important mechanism to minimize errors during DNA synthesis. The polymerase-associated proofreading exonuclease was ...

The Msh2-Msh6 heterodimer plays a key role in the repair of mispaired bases in DNA. Critical to its role in mismatch repair is the ATPase activity that resides within each subunit. Here we show that both subunits can simultaneously bind ATP and identify the Msh6 subunit as containing the high-affinity ATP binding site and Msh2 as containing a high-affinity ADP binding site. Stable binding of ATP to Msh6 causes decreased affinity of Msh2 for ADP, and binding to mispaired DNA stabilized the binding of ATP to Msh6. Our results support a model in which mispair binding encourages a dual-occupancy state with ATP bound to Msh6 and Msh2; this state supports hydrolysis-independent sliding along DNA.