The binding of hMutSα to mismatches and its interaction with hMutL heterodimers on DNA was investigated by a method that enabled the use of short incubation times and cell extracts, which allowed the mimicking of biological binding conditions more closely. Binding of all proteins in this assay was confirmed to be DNA dependent, and consecutive washing steps of increasing stringency allowed the existence of mismatch-specific binding of hMutSα to be proved. Furthermore, the mismatch binding ability of hMutSα was shown to be altered at the same ATP concentration as reported previously, confirming the reliability of the method. The higher salt resistance exhibited by hMutSα when bound to heteroduplexes likely mirrors the tighter and more stable interaction of MutS proteins with mismatched DNA that has previously been visualized in the crystal structure of bacterial MutS and heteroduplex DNA (6
). The data from this study also show that hMutLα binds single-stranded DNA, providing evidence that the function of human MutLα is similar to bacterial MutL, which also preferentially binds this substrate (21
). Binding of hMutLα to single-stranded DNA was not, however, enhanced by the non-hydrolyzable ATP analog AMP-PNP, possibly reflecting a lower susceptibility to these nucleotides under the assay conditions used.
ATP has been shown to reduce the affinity of MutS and its eukaryotic homologs for mismatches (12
), which was interpreted as a complete loss of mismatch binding ability. Recent studies with bacterial MutS, however, indicated that ATP binding and mismatch binding by MutS may not be mutually exclusive (18
). The present study confirms that the affinity (in terms of salt resistance) of hMutSα for homo- and heteroduplexes is markedly reduced in the presence of ATP, but also indicates that binding to homoduplexes may be more affected than binding to heteroduplexes. With regard to the current models of mismatch repair, this observation is in accordance with the DNA bending model (18
). In this case, the reduced salt resistance of the hMutSα–mismatch complex in the presence of 250 µM ATP may represent the state of verification that hMutSα would enter after binding ATP.
Furthermore, hMutSα was found to interact with hMutLα and hMutLβ on DNA in the presence of ATP. This interaction occurred only on 81mer and not on 32mer DNA substrates, reflecting a DNA length dependence of complex formation that has recently also been observed for bacterial (31
) and human complexes using SPRS and gel shift assays (32
). The finding that hMutLα has an intrinsic affinity for DNA supports the notion that both heterodimers may stay in contact with DNA in the complex. Because hMutSα covers ~25 bp of DNA (36
), the 32mer substrate may not be long enough for interaction.
According to recent reports, the involvement of hMutLβ in mismatch repair remains controversial. hMutLβ was suggested to participate in human mismatch repair due to a hPMS1
mutation found in a patient with hereditary non-polyposis colorectal cancer (HNPCC), a cancer predisposition syndrome associated with mutations in mismatch repair genes (9
). Furthermore, PMS1
-deficient mouse fibroblasts exhibit microsatellite instability, a phenotypic marker of deficient mismatch repair (10
). However, hMutLβ is unable to confer mismatch repair proficiency on hMLH1-deficient extracts, which is possible with hMutLα (8
). The present study shows that hMutLβ, although being 10 times less abundant than hMutLα in HeLa nuclear extracts (11
), is efficiently recruited by hMutSα. The results also show that hMutLα is the favored partner for interaction with hMutSα at low ATP concentrations. Taken together, the results show that hMutLβ participates efficiently in mismatch repair protein complexes. It may exert a supportive function in the repair process after initial formation of the hMutSα and hMutLα complex. Further studies are necessary to elucidate the precise contribution of MutLβ to the mismatch repair process.
Under the conditions used in the present study, hMutSα interacted with hMutL heterodimers on homoduplexes and heteroduplexes, although complex formation was enhanced on heteroduplexes, which is consistent with earlier findings (28
). In contrast, a recent report found complex formation to be mismatch dependent using SPRS and that different hMutSα–hMutLα complexes arose on homo- and heteroduplex DNA in gel shift assays (32
), showing that the experimental procedure influences the mode of interaction. The ability to interact on homoduplex DNA may be explained by the sliding clamp model (17
) as well as the translocation model (13
), since both predict an interaction of hMutSα with other components of the mismatch repair machinery after ATP-induced movement from the mismatch to homoduplex DNA.
A recent report showed the dynamic nature of human MutSα–MutLα complexes on DNA, and the complexes were found to dissociate from the substrate DNA on poly[d(I-C)] challenge in gel shift assays when the DNA was not blocked at both ends (32
). Although poly[d(I-C)] was present in our experiments (except for those with purified proteins) and the DNA substrates were generally blocked at only one end, hMutSα–hMutL complexes remained detectable. A possible explanation is that an equilibrium may be established between formation of hMutSα–hMutL complexes on DNA and translocation of these complexes off the oligoduplex during incubation and that only the fraction bound at the moment of elution is subsequently detected. Alternatively, under the assay conditions used, translocation off the oligoduplex may be blocked by other means than a second end block. The complex may, for example, be tethered to the substrate by additional proteins. This would also account for the weaker signals seen in experiments with purified proteins compared with cell extracts.
hMutSα contains two asymmetrical ATPase sites and it is possible that one ATPase predominantly confers interaction with hMutL proteins after mismatch recognition. The finding that the hMSH6 ATPase mutant in MT1 cells efficiently recruited hMutL heterodimers suggests that hMSH2 predominantly initiates the interaction (which has also been proposed based on considerations of the X-ray structure of bacterial MutS on a mismatch; 6
), while the ATP function of hMSH6 (the subunit that directly contacts the mismatch) may promote subsequent processes, like mismatch verification in the DNA bending model. However, further studies are necessary to elucidate the different contributions of the two ATPases to these processes.
In conclusion, DNA-coupled magnetic beads provide a suitable tool for investigating protein–DNA interactions and formation of mismatch repair protein complexes. The affinity of hMutSα for homoduplexes is shown to be more reduced by ATP than the affinity for heteroduplexes, supporting the recently suggested DNA bending model of mismatch repair (18
). Furthermore, hMutSα is shown to interact with hMutLα as well as hMutLβ on DNA. This interaction requires ATP and occurs only on long DNA substrates, confirming the DNA length dependence of complex formation reported earlier. Although hMutLβ is efficiently recruited by hMutSα, hMutLα seems to be the preferred partner in the initial interaction and it seems reasonable that hMutLβ exerts an auxiliary function in the mismatch repair process.