Mispair-bound human MutS–MutL complex triggers DNA incisions and activates mismatch repair

DNA mismatch repair (MMR) relies on MutS and MutL ATPases for mismatch recognition and strand-specific nuclease recruitment to remove mispaired bases in daughter strands. However, whether the MutS–MutL complex coordinates MMR by ATP-dependent sliding on DNA or protein–protein interactions between the mismatch and strand discrimination signal is ambiguous. Using functional MMR assays and systems preventing proteins from sliding, we show that sliding of human MutSα is required not for MMR initiation, but for final mismatch removal. MutSα recruits MutLα to form a mismatch-bound complex, which initiates MMR by nicking the daughter strand 5′ to the mismatch. Exonuclease 1 (Exo1) is then recruited to the nick and conducts 5′ → 3′ excision. ATP-dependent MutSα dissociation from the mismatch is necessary for Exo1 to remove the mispaired base when the excision reaches the mismatch. Therefore, our study has resolved a long-standing puzzle, and provided new insights into the mechanism of MMR initiation and mispair removal.

Exo1 Independent DNA Mismatch Repair Pathway in Hematopoiesis.

Abstract 2526 Poster Board II-503 The DNA mismatch repair pathway (MMR) is a fundamental process in cells that functions to correct mispaired bases and insertion/deletion loops caused by errors in replication. Failure in MMR can lead to the accumulation of mutations and carcinogenesis, notably hereditary nonpolyposis colorectal cancer (HNPCC). In hematopoiesis, loss of MMR results in methylating agent resistance and an HSC repopulation defect. Our research focused on the significance of Exonuclease 1, the enzyme responsible for excising mispaired bases in MMR. Interestingly Exo1−/− mice display a much milder pathogenic phenotype when compared to the MMR deficient MSH2−/− mice; characterized by lower mutation rates, lower levels of microsatellite instability, and in humans no association with HNPCC. These findings led us to hypothesize that the limited phenotype observed in Exo1−/− mice is due to (a) complementation through another exonuclease occurs which restores the proficiency of the MMR pathway or (b) an alternative pathway exists which allows for exonuclease independent repair. To test this hypothesis we derived primary MEFs from WT, Exo1−/− and MSH2−/− mice and performed temozolomide sensitivity assays. We observed that Exo1−/− MEFs are similarly sensitive as WT cells when treated with the drug, confirming proficient MMR, while MSH2−/− MEFs display a decreased sensitivity. Additionally, comet assays with the same cell populations show a persistence of DNA single strand breaks in the Exo1−/− and WT cells 24 hours post temozolomide treatment, consistent with proficient MMR activity, while MSH2−/− MEFs show no such damage. To confirm functional MMR in hematopoiesis of Exo1−/− mice we conducted a competitive repopulation study. We found that Exo1−/− and WT marrow engraft at similar levels 8 and 16 weeks post transplantation. We subsequently treated these mice with 80 mg/kg temozolomide to determine if Exo1−/− marrow progenitors would confer a competitive survival advantage post treatment. Consistent with our hypothesis we found that the ratio of marrow cells remained approximately 50:50 (Exo1:WT) in contrast to data obtained from MSH2−/− mice in which the MSH2−/− cells display a strong 95:5 (MSH2:WT) advantage post drug treatment. This strongly suggests functional MMR in the Exo1 deficient mice. We next coimmunoprecipitated the MMR complex by pulling down Mlh1 in Exo1−/− and WT MEFs. Exo1, a key exonuclease in mismatch repair, does not modulate hematopoietic function as do the other MMR proteins and appears to be complemented by an as yet undefined exonuclease. Disclosures: No relevant conflicts of interest to declare.

N.m.r. determination of the solution conformation and dynamics of the A.G mismatch in the d(CGCAAATTGGCG)2 dodecamer

A.G base-paired mismatches that occur during replication are among the most difficult to detect by repair enzymes. Such purine.purine mispairs can exist in two conformations, one of which is stabilized by protons [Gao & Patel (1988) J. Am. Chem. Soc. 110, 5178-5182]. We have undertaken a 1H-n.m.r. and 31P-n.m.r. study of the mismatched dodecamer d(CGCAAATTGGCG)2 as a function of both temperature and pH to determine the conformational features of the A.G mismatch. At pH greater than 7 the mispaired bases are each in the anti conformation and are stacked in the B-like helix. As the pH is decreased, a second conformation becomes populated (apparent pKa approx. 5.9) with concomitant changes in the chemical shifts of protons of the mispaired bases and their nearest neighbours. Data from two-dimensional nuclear-Overhauser-enhancement spectroscopy show unequivocally that, at low pH, the dominant conformation is one in which the mismatched G residues are in the syn conformation and are hydrogen-bonded to the A residues that remain in the anti conformation. Residues not adjacent to the A.G sites are almost unaffected by the transition or the mispairing, suggesting considerable local flexibility of the unconstrained duplexes. Despite the bulging of the mispaired bases, the conformation of the A(anti).G(anti) duplex is very similar to the native dodecamer, whereas the AH+(anti).G(syn) duplex shows a greater variation in the backbone conformation at the mismatched site. According to the chemical shifts, the duplex retains twofold symmetry in solution. The equilibrium between the syn and anti conformations of G9/G21 is strongly dependent on pH, but only weakly dependent on temperature (delta H approx. 16 kJ.mol-1). The first-order rate constant for the transition is approx. 9 s-1 at 283 K and approx. 60 s-1 at 298 K, with an activation enthalpy of approx. 100 kJ.mol-1. The stabilization of the A(anti).G(syn) conformation by protons is consistent with models invoking N1 protonation of adenine. Using the derived glycosidic torsion angles we have used restrained molecular dynamics to build models of the neutral and protonated d(CGCAAATTGGCG)2 oligomers. The results confirm that the A(anti).G(anti) and AH+(anti).G(syn) conformations are favoured at high pH and low pH respectively, in accord with n.m.r. and single-crystal X-ray data.