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

Abstract

In early reactions of DNA mismatch repair, MutS recognizes mismatched bases and activates MutL endonuclease to incise the error-containing strand of the duplex. DNA sliding clamp is responsible for directing the MutL-dependent nicking to the newly synthesized/error-containing strand. In Bacillus subtilis MutL, the ␤-clamp-interacting motif (␤ motif) of the C-terminal domain (CTD) is essential for both in vitro direct interaction with ␤-clamp and in vivo repair activity. A large cluster of negatively charged residues on the B. subtilis MutL CTD prevents nonspecific DNA binding until ␤ clamp interaction neutralizes the negative charge. We found that there are some bacterial phyla whose MutL endonucleases lack the ␤ motif. For example, the region corresponding to the ␤ motif is completely missing in Aquifex aeolicus MutL, and critical amino acid residues in the ␤ motif are not conserved in Thermus thermophilus MutL. We then revealed the 1.35 Å-resolution crystal structure of A. aeolicus MutL CTD, which lacks the ␤ motif but retains the metalbinding site for the endonuclease activity. Importantly, there was no negatively charged cluster on its surface. It was confirmed that CTDs of ␤ motif-lacking MutLs, A. aeolicus MutL and T. thermophilus MutL, efficiently incise DNA even in the absence of ␤-clamp and that ␤-clamp shows no detectable enhancing effect on their activity. In contrast, CTD of Streptococcus mutans, a ␤ motif-containing MutL, required ␤-clamp for the digestion of DNA. We propose that MutL endonucleases are divided into three subfamilies on the basis of their structural features and dependence on ␤-clamp.

The mutagen and carcinogen cadmium is a high-affinity inhibitor of the zinc-dependent MutLα endonuclease

“…This suggests that activation by millimolar Mn 2+ is the result of substitution for one or both endogenous zinc ions and that this effect can be reversed by low concentrations of exogenous Zn 2+ . The endonuclease motifs described above are found in many but not all bacterial MutL proteins ( 3 , 30 ), where they are also believed to comprise Zn 2+ binding endonuclease active sites that are subject to Mn 2+ activation ( 31 ). NMR analysis of the Aquifex aeolicus MutL CTD has shown that Mn 2+ binds in the proximity of the DQHA(X) 2 E(X) 4 E and CPHGRP zinc coordination motifs ( 32 ), which is consistent with our findings based on functional assays.…”

Multiple zinc ions maintain the open conformation of the catalytic site in the DNA mismatch repair endonuclease MutL from Aquifex aeolicus

“…Previously, we reported the crystal structure of the aqMutL CTD complexed with three cadmium ions . Here, in order to reveal the crystal structure with zinc ions, we incubated the aqMutL CTD in a buffer containing 0.5 m m zinc acetate, concentrated the protein, and created the crystal in the presence of 5 m m zinc ions.…”

“…Zinc ions are shown as cyan spheres. (B) The crystal structure of the aqMutL CTD monomer with three cadmium ions ( PDB ID : 5B42) . Cadmium ions are shown as orange spheres.…”

“…Structure of the aqMutL CTD with three zinc ions Previously, we reported the crystal structure of the aqMutL CTD complexed with three cadmium ions [23].…”

“…In a previous study, we reported the crystal structure of the CTD of aqMutL complexed with three cadmium ions . The first and second binding sites of cadmium ions were located at the positions that were exactly the same as those for the two zinc‐binding sites in the B. subtilis MutL CTD.…”