Crystal Structure of MutS2 Endonuclease Domain and the Mechanism of Homologous Recombination Suppression

Abstract

DNA recombination events need to be strictly regulated, because an increase in the recombinational frequency causes unfavorable alteration of genetic information. Recent studies revealed the existence of a novel anti-recombination enzyme, MutS2. However, the mechanism by which MutS2 inhibits homologous recombination has been unknown. Previously, we found that Thermus thermophilus MutS2 (ttMutS2) harbors an endonuclease activity and that this activity is confined to the C-terminal domain, whose amino acid sequence is widely conserved in a variety of proteins with unknown function from almost all organisms ranging from bacteria to man. In this study, we determined the crystal structure of the ttMutS2 endonuclease domain at 1.7-Å resolution, which resembles the structure of the DNase I-like catalytic domain of Escherichia coli RNase E, a sequence-nonspecific endonuclease. The N-terminal domain of ttMutS2, however, recognized branched DNA structures, including the Holliday junction and D-loop structure, a primary intermediate in homologous recombination. The full-length of ttMutS2 digested the branched DNA structures at the junction. These results indicate that ttMutS2 suppresses homologous recombination through a novel mechanism involving resolution of early intermediates.Homologous recombination is required for a variety of DNA transactions such as DNA repair, the rescue of stalled replication forks, and the creation of genetic diversity (1-4). The reaction begins with the introduction of a double strand break in the homologous region of the donor strand (5-7). The ends of the strand are resected by exonucleases to generate termini with 3Ј-overhangs. Then, RecA protein (Rad51 in eukaryotes) recognizes the single-stranded region of the DNA and catalyzes the invasion into the target double-stranded DNA to generate the D-loop structure. The sequential DNA synthesis and ligation yield a general intermediate called the Holliday junction. Several junction-specific endonucleases resolve the junction, and DNA ligase reseals the nicks to complete the process of homologous recombination. This homologous recombination process is utilized not only for the recovery but also for the alteration of genetic information.To avoid unfavorable alteration of genetic information, organisms are equipped with a series of enzymes that not only promote but also suppress homologous recombination (4). Recent studies have shown that bacterial and plant MutS2 proteins are candidate anti-recombination enzymes (8, 9). MutS2 is a paralogue of Escherichia coli MutS, which recognizes a mismatched base pair and induces the mismatch repair (MMR) 2 pathway (Fig. 1). Proteins homologous to E. coli MutS are widely distributed and classified into two subfamilies, MutSI and MutSII, on the basis of amino acid sequence comparison (10). The former is responsible for MMR and includes bacterial MutS and eukaryotic MSH2, MSH3, and MSH6 (11-12). The latter, MutSII, is expected to not be involved in MMR but in recombinational events, and includes bacterial MutS2 a...

MutS2 Promotes Homologous Recombination in Bacillus subtilis

“…To our knowledge, our observation of MMC sensitivity is the first phenotype shown for a ΔmutS2 deletion in B. subtilis. A previous study of Thermus thermophilus mutS2 found that deletion of mutS2 resulted in resistance to MMC treatment relative to the wild type, consistent with the inhibitory effect of TtMutS2 on recombination (22). Given that deletion of B. subtilis mutS2 has the opposite effect of TtΔmutS2, these results suggest that B. subtilis MutS2 promotes homologous recombination, a novel result for a bacterial MutS2 protein.…”

“…The function of B. subtilis MutS2 (BsMutS2) in genome maintenance is unknown. We initially hypothesized that B. subtilis MutS2 participated in antirecombination, as demonstrated for H. pylori and T. thermophilus MutS2 (19,22). Instead, here we report that MutS2 promotes homologous recombination in B. subtilis.…”

“…H. pylori and T. thermophilus strains lacking mutS2 are more efficiently transformed with DNA, and the T. thermophilus ΔmutS2 strain is more resistant than the wild type (WT) to mitomycin C (MMC), an antibiotic that damages DNA and requires homologous recombination for repair (19,22). Biochemical studies have shown that HpMutS2 and T. thermophilus MutS2 (TtMutS2) both have ATPase activity that is stimulated by recombination intermediates, and TtMutS2 has endonuclease activity toward recombination intermediates, including Holliday junctions (19,22). These data support the hypothesis that MutS2 proteins suppress homologous recombination in bacteria.…”

“…MutS2 is composed of an N-terminal region involved in DNA binding, followed by a central ABC ATPase domain, which also participates in dimerization (21,22). The MutS2 C-terminal domain contains the small MutS-related (Smr) region, harboring a DNase I-like endonuclease domain (22).…”

Two new subfamilies of DNA mismatch repair proteins (MutS) specifically abundant in the marine environment

“…In this study, we use the following naming of the MutS subfamilies, which is adapted from the recent study by Lin et al (Lin et al, 2007). In total, 12 subfamilies were previously described to compose the MutS family: 'MutS1/ MSH1' including E. coli MutS and the mitochondria-targeted fungal MutS homolog 1 (MSH1); 'MutS2', known to inhibit recombination in H. pylori (Pinto et al, 2005) and to possess a C-terminal endonuclease domain called the small MutS-related (Smr) domain (Moreira and Philippe, 1999;Fukui et al, 2008); 'MSH2', 'MSH3', 'MSH4', 'MSH5' and 'MSH6/7', found in most eukaryotes (with the exception of MSH7 being a plant-specific paralogous group of MSH6 (Wu et al, 2003)); another plantspecific MSH1 (called 'plt-MSH1' hereafter) with the GIY-YIG endonuclease domain at their C-terminus (Abdelnoor et al, 2006); 'MutS3', 'MutS4' and 'MutS5', recently described but functionally uncharacterized prokaryotic homologs (Lin et al, 2007), and the above mentioned 'MutS7' subfamily represented by the Mimivirus MutS homolog.…”