MutS stimulates the endonuclease activity of MutL in an ATP‐hydrolysis‐dependent manner

Abstract

In the initial steps of DNA mismatch repair, MutS recognizes a mismatched base and recruits the latent endonuclease MutL onto the mismatch-containing DNA in concert with other proteins. MutL then cleaves the error-containing strand to introduce an entry point for the downstream excision reaction. Because MutL has no intrinsic ability to recognize a mismatch and discriminate between newly synthesized and template strands, the endonuclease activity of MutL is strictly regulated by ATP-binding in order to avoid nonspecific degradation of the genomic DNA. However, the activation mechanism for its endonuclease activity remains unclear. In this study, we found that the coexistence of a mismatch, ATP and MutS unlocks the ATP-binding-dependent suppression of MutL endonuclease activity. Interestingly, ATPase-deficient mutants of MutS were unable to activate MutL. Furthermore, wild-type MutS activated ATPase-deficient mutants of MutL less efficiently than wild-type MutL. We concluded that ATP hydrolysis by MutS and MutL is involved in the mismatch-dependent activation of MutL endonuclease activity.

Structural Features and Functional Dependency on β-Clamp Define Distinct Subfamilies of Bacterial Mismatch Repair Endonuclease MutL

“…This notion is also supported by previously reported in vitro partial reconstitution system of T. thermophilus MMR, where ␤-clamp has no effect on the strand incision (43). To unveil the mechanism, biochemical properties of clamp-independent MutL endonuclease should be further examined by experiments, such as investigating the nucleotide-sequence specificity, binding partner, and response to ATP.…”

“…MutL endonucleases from Deinococcus-Thermus phylum retain the regulatory subdomain in their CTDs; nevertheless, they have no sequence satisfying the criteria for the clamp-interacting motif. Our previous study showed that the endonuclease activity of ttMutL was indeed required for in vivo MMR activity but that ␤-clamp and clamp-loader were not able to direct the ttMutL-dependent incision to the discontinuous strand in the in vitro reconstituted system (43). As shown in Fig.…”

“…aqMutL, aqMutL CTD, ttMutL, His-tagged ttMutL CTD, and T. thermophilus ␤-clamp were prepared as previously described (19,43,50). Mutants of aqMutL, aqMutL CTD, ttMutL, and Histagged ttMutL CTD were prepared by the same procedure as for wild type proteins.…”

“…1). We previously reported a partial in vitro reconstituted MMR system by using proteins from Thermus thermophilus, where incision by MutL was mismatch-, MutS-, and ATP-dependent (43). However, T. thermophilus MutL incised both strands of the duplex despite the existence of strand discontinuity, ␤-clamp, and clamp loader.…”

Mismatch Repair during Homologous and Homeologous Recombination

“…The MLH/PMS endonuclease appears more efficient in the presence of manganesedivalent cation and can also be stimulated by zinc (Kadyrov et al 2006(Kadyrov et al , 2007Pillon et al 2010). Importantly, the Thermus thermophilus homologs were used to show that the TtMutL ATP-dependent endonuclease is activated only on its association with ATP-bound TtMutS sliding clamps (Shimada et al 2013).…”