The absolute magnitude of ATPase activity of MutS reported in the previous publications shows widely dispersed values (2–7 min?1, monomer) depending on the experimental conditions and assay methods [5], [12], [14] and [25]. Most of the previous kcat values were obtained from basically initial velocity method (using γ-32P-ATP). The apparent large increase of ATPase activity of MutS in the presence of homo- and heteroduplex DNAs, in fact, results from the inclusion of initial burst ATP hydrolysis rate. The isotopic assay method may be most appropriate for the small sample volume since it does not provide the true steady-state ATP hydrolysis rate. The ATPase activity of MutS in the absence of DNA in this work ( Fig. S2) is similar to the previously reported values [25].The most striking feature found in this work is the enhancement of the steady-state ATPase activity of trapped MutS upon addition of MutL in the presence of double blocked end G–T heteroduplex DNAs. To look for the effect of ATPase activity of MutL on the trapped MutS sliding clamps, we have deliberately chosen and prepared four different mutant MutL
R-115777 which are defective either in ATP hydrolysis (E29A and R261H) or in ATP binding (D58A), or in DNA binding (R266E) properties, and investigated the effect of these mutant MutLs on the steady-state ATPase activity of the trapped MutS. While the mutant MutL proteins defective in ATPase activity do not influence the steady-state ATPase activity of trapped MutS on double blocked end G–T heteroduplexes, the significant enhancement of MutS ATPase activity by wild-type and R266E MutL (Fig. 3) implicitly suggests that ATP hydrolysis by MutL triggers conformational change of the trapped MutS and leads to dissociation of the MutS sliding clamp. Based on the results from total internal reflectance (TIR) and gel shift experiments, Acharya and coworkers have proposed that MutS sliding clamp release occurs by intrinsic MutS ATP hydrolysis that is accelerated by MutL [5]. They claimed that the MutS clamp-unloading function occurs independently of ATP binding or hydrolysis by MutL. Although, the role of MutL for catalyzing dissociation of the MutS sliding clamp is consistent with our results, the results from mutant MutL
proteins in this work support that the ATPase activity of MutL is critical for the unloading of the MutS sliding clamp.Based on the results observed in this work and previous reports from others [9], [10] and [11], the sliding clamp model for the double blocked end heteroduplex in MMR has been modified to accommodate the role of MutL ATPase activity (Fig. 4). In this work, we did not observe any change in the suppressed ATPase activities of MutS in the presence of double blocked end G–T heteroduplexes until MutL with ATPase activity is added. Thus, a direct dissociation channel for the ATP-bound MutS sliding clamps is not considered as one of the major channel, and ATP hydrolysis of MutL provokes dissociation of MutS sliding clamps either through conformational change or hydrolysis of bound ATP.Figure 4. Model of interactions of ATP-bound MutS and MutL on double blocked end (G–T) heteroduplex. The ATP-bound MutS sliding clamps recruit ATP-bound MutL and ATP hydroysis of MutL induces conformation change of the MutS sliding clamps to dissociate from the double blocked end heteroduplex DNA.Figure optionsDownload full-size imageDownload as PowerPoint slideAcknowledgmentsThis work was supported by grants from the KOSEF through the Center for Integrated Molecular System at POSTECH (R11-2000-070-04005-0) and the Korea Research Foundation Grant (KRF-2008-314-C0021
cool .Appendix A. Supplementary dataSupplementary data. Help with DOC filesOptionsDownload file (1178 K).
