Contribution of Human Mlh1 and Pms2 ATPase Activities to DNA Mismatch Repair

Abstract

MutL␣, a heterodimer composed of Mlh1 and Pms2, is the major MutL activity in mammalian DNA mismatch repair. Highly conserved motifs in the N termini of both subunits predict that the protein is an ATPase. To study the significance of these motifs to mismatch repair, we have expressed in insect cells wild type human MutL␣ and forms altered in conserved glutamic acid residues, predicted to catalyze ATP hydrolysis of Mlh1, Pms2, or both. Using an in vitro assay, we showed that MutL␣ proteins altered in either glutamic acid residue were each partially defective in mismatch repair, whereas the double mutant showed no detectable mismatch repair. Neither strand specificity nor directionality of repair was affected in the single mutant proteins. Limited proteolysis studies of MutL␣ demonstrated that both Mlh1 and Pms2 N-terminal domains undergo ATP-induced conformational changes, but the extent of the conformational change for Mlh1 was more apparent than for Pms2. Furthermore, Mlh1 was protected at lower ATP concentrations than Pms2, suggesting Mlh1 binds ATP with higher affinity. These findings imply that ATP hydrolysis is required for MutL␣ activity in mismatch repair and that this activity is associated with differential conformational changes in Mlh1 and Pms2. Mismatch repair (MMR)1 helps to protect the genome from replication errors caused by DNA polymerases. Its pivotal role as caretaker of genome stability is exemplified by HNPCC, a hereditary predisposition to colon, rectal, and other cancers associated with germ line mutations in MMR (1).In Escherichia coli, the major players in the pathway are known to the extent that a full in vitro mismatch repair was reconstituted from purified components (2). A MutS homodimer recognizes and binds a mismatch and recruits a MutL homodimer to form an ATP-dependent ternary complex with DNA (3). MutL recruits and activates MutH (4), an endonuclease that introduces a specific nick on hemimethylated GATC sites, thus providing the strand discrimination signal required to ensure repair of the nascent strand. MutL also activates UvrD (5), a helicase that unwinds the duplex DNA from the nick, providing a single strand DNA substrate for an exonuclease. The repair pathway is bidirectional in that repair can initiate from GATC sites located either 5Ј or 3Ј to the mismatch (6). As a result, of the four exonucleases that have been found to participate in the pathway, two have 5Ј to 3Ј polarity, and the others have 3Јto 5Ј polarity (7). The single strand gap formed by the exonuclease is filled in by DNA polymerase III, and the nick is sealed by DNA ligase.The pathway in eukaryotes is less understood than in E. coli (reviewed in Refs. 8 and 9). Instead of one MutS, there are three MutS-like proteins that are involved in mutation avoidance, Msh2, Msh3, and Msh6. Msh2 and Msh6 associate to form MutS␣, the major MutS activity in MMR (reviewed in Ref. 9). Illustrating a similar level of complexity, there are four MutL homologs in mammals: Mlh1, Pms2 (closest to Pms1 in yeast), Pms1, and Mlh3....

Modulation of microRNA processing by mismatch repair protein MutLα

“…These observations indicate that ATP hydrolysis by MutLα is essential for its ability to stimulate miRNA processing. This idea was further examined using MMR-deficient MutLα proteins MutLαEA, MutLαF99L and MutLαE705K [29,34,35]. Our results show that MutLαEA and MutLαF99L are proficient, but MutLαE705K is deficient in pri-miRNA binding ( Figure 4D).…”

“…As shown in Figure 4D, MutLα indeed specifically shifted 32 P-labeled pri-miR-422a even in the presence of excess, unlabeled competitor tRNA. Pri-miR-422a-binding reactions were also carried out with several MMR-deficient MutLα mutants, MutLαEA (MLH1 E34A -PMS2 E41A ) [33,34], MutLαF99L (MLH1 F99L -PMS2) [29] and MutLαE705K (MLH1-PMS2 E705K ) [35]. While MutLαEA ( Figure 4D, lanes 6-7) and MutLαF99L ( Figure 4D, lanes 8-9) bound pri-miR-422a as efficiently as the WT MutLα, MutLαE705K failed to interact with pri-miR-422a ( Figure 4D, lanes 10-11).…”

“…npg for its function in MMR [33,34]. We therefore examined whether ATP hydrolysis by MutLα is essential for its ability to promote pri-miRNA processing.…”

“…All MutL heterodimers were expressed in pFastBac Dual vector and purified as described [34,47]. The purity of all recombinant proteins was assessed by SDS-PAGE and protein concentrations were determined by Bradford assay.…”

Variability in the clinical phenotype among families with HNPCC—The potential importance of the location of the mutation in the gene

“…Also, the MSH6 protein has the MSH2 binding domains and the ATP binding region, and the PMS2 protein has the MLH1 and ATP binding domains. Some of these regions have in fact also been shown to be critical for the function of mismatch repair 14 and mutations in these specific areas could particularly reduce the interaction efficiency and/or significantly interfere with the mismatch repair process. 15 In addition, it has also recently been shown 16 that the MSH2 protein interacts with the estrogen receptor-a through the MSH6/MSH3 binding domain, which could account for the relationship between some MSH2 and MSH6 mutations, and estrogen dependent cancers such as endometrial/ovarian cancers.…”