Abstract
MutL is a multi-domain protein comprising an N-terminal ATPase domain (NTD) and C-terminal dimerization domain (CTD), connected with flexible linker regions, that plays a key role in DNA mismatch repair. To expand understanding of the regulation mechanism underlying MutL endonuclease activity, our NMR-based study investigated interactions between the CTD of MutL, derived from the hyperthermophilic bacterium Aquifex aeolicus (aqMutL-CTD), and putative binding molecules. Chemical shift perturbation analysis with the model structure of aqMutL-CTD and circular dichroism results revealed that tight Zn2+ binding increased thermal stability without changing secondary structures to function at high temperatures. Peak intensity analysis exploiting the paramagnetic relaxation enhancement effect indicated the binding site for Mn2+, which shared binding sites for Zn2+. The coexistence of these two metal ions appears to be important for the function of MutL. Chemical shift perturbation analysis revealed a novel ATP binding site in aqMutL-CTD. A docking simulation incorporating the chemical shift perturbation data provided a putative scheme for the intermolecular interactions between aqMutL-CTD and ATP. We proposed a simple and understandable mechanical model for the regulation of MutL endonuclease activity in MMR based on the relative concentrations of ATP and CTD through ATP binding-regulated interdomain interactions between CTD and NTD.
Highlights
DNA mismatch repair (MMR) is the process by which postreplicative base pair errors are rectified, thereby enhancing the fidelity of DNA replication 100–1000 fold [1]
While E. coli MMR possesses MutH endonuclease, which cleaves daughter strands at hemi-methylated GATC sites, eukaryotes and most bacteria lack MutH; the counterpart of MutH in eukaryotic and eubacteria species was a topic for speculation until the latent endonuclease activities of human and yeast MutLa were demonstrated in the presence of divalent metal ions such as Mn2+ [8,9]
The homology model was generated under the constraints of the secondary structures predicted by TALOS+ [28], estimating dihedral angles based on the assigned chemical shift information of CO, Ca, Cb, HN, and 15N of aqMutL-C-terminal dimerization domain (CTD) (Figure 1C)
Summary
DNA mismatch repair (MMR) is the process by which postreplicative base pair errors are rectified, thereby enhancing the fidelity of DNA replication 100–1000 fold [1]. MMR starts from the recognition of mismatched base pairs by MutS, and is followed by the recruitment of MutL by the mismatch:MutS complex. The mismatch:MutS:MutL complex has been shown to initiate downstream repair machinery in both E. coli and eukaryotes, in which newly synthesized DNA strands are nicked at either side of the mismatch [6,7]. The mechanisms underlying daughter strand recognition and incision are decisively different between E. coli and eukaryotes. While E. coli MMR possesses MutH endonuclease, which cleaves daughter strands at hemi-methylated GATC sites, eukaryotes and most bacteria lack MutH; the counterpart of MutH in eukaryotic and eubacteria species was a topic for speculation until the latent endonuclease activities of human and yeast MutLa were demonstrated in the presence of divalent metal ions such as Mn2+ [8,9]
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