title: 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 β-clamp-interacting motif (β motif) of the C-terminal domain (CTD) is essential for both in vitro direct interaction with β-clamp and in vivo repair activity. A large cluster of negatively charged residues on the B. subtilis MutL CTD prevents nonspecific DNA binding until β clamp interaction neutralizes the negative charge. We found that there are some bacterial phyla whose MutL endonucleases lack the β motif. For example, the region corresponding to the β motif is completely missing in Aquifex aeolicus MutL, and critical amino acid residues in the β 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 β motif but retains the metal-binding site for the endonuclease activity. Importantly, there was no negatively charged cluster on its surface. It was confirmed that CTDs of β motif-lacking MutLs, A. aeolicus MutL and T. thermophilus MutL, efficiently incise DNA even in the absence of β-clamp and that β-clamp shows no detectable enhancing effect on their activity. In contrast, CTD of Streptococcus mutans, a β motif-containing MutL, required β-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 β-clamp.
authors.affiliation: From the Department of Biochemistry, Osaka Medical College, 2-7, Daigakumachi, Takatsuki, Osaka 569-8686 and k.fukui@osaka-med.ac.jp.
authors.forename: Kenji
authors.initials: K
authors.lastname: Fukui
authors.affiliation: Japan Synchrotron Radiation Research Institute (JASRI), SPring-8, Kouto, Sayo, Hyogo 679-5198, Japan.
authors.forename: Seiki
authors.initials: S
authors.lastname: Baba
authors.affiliation: Japan Synchrotron Radiation Research Institute (JASRI), SPring-8, Kouto, Sayo, Hyogo 679-5198, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Kumasaka
authors.affiliation: From the Department of Biochemistry, Osaka Medical College, 2-7, Daigakumachi, Takatsuki, Osaka 569-8686 and med013@osaka-med.ac.jp.
authors.forename: Takato
authors.initials: T
authors.lastname: Yano
mesh_terms: D020816:Amino Acid Motifs; D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry*; D018360:Crystallography, X-Ray; D000070956:MutL Proteins / Q000737:chemistry*; D000072417:Protein Domains; D013295:Streptococcus mutans / Q000201:enzymology*; D016963:Thermus thermophilus / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D000070956:MutL Proteins
keywords: DNA binding protein; DNA mismatch repair; MutL; bacteria; clamp; endonuclease; x-ray crystallography