Nuclease activity of the MutS homologue MutS2 from Thermus thermophilus is confined to the Smr domain

Abstract

MutS homologues are highly conserved enzymes engaged in DNA mismatch repair (MMR), meiotic recombination and other DNA modifications. Genome sequencing projects have revealed that bacteria and plants possess a MutS homologue, MutS2. MutS2 lacks the mismatch-recognition domain of MutS, but contains an extra C-terminal region called the small MutS-related (Smr) domain. Sequences homologous to the Smr domain are annotated as ‘proteins of unknown function’ in various organisms ranging from bacteria to human. Although recent in vivo studies indicate that MutS2 plays an important role in recombinational events, there had been only limited characterization of the biochemical function of MutS2 and the Smr domain. We previously established that Thermus thermophilus MutS2 (ttMutS2) possesses endonuclease activity. In this study, we report that a Smr-deleted ttMutS2 mutant retains the dimerization, ATPase and DNA-binding activities, but has no endonuclease activity. Furthermore, the Smr domain alone was stable and functional in binding and incising DNA. It is noteworthy that an endonuclease activity is associated with a MutS homologue, which is generally thought to recognize specific DNA structures.

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

“…On the other hand, an expression of the N60-ttmutS2 mutant did not give rise to a serious decrease in the survival ratio. We have reported that the N60-ttMutS2 mutant binds to dsDNA as tightly as wild-type ttMutS2, however, N60-ttMutS2 does not retain the endonuclease activity (17). The endonuclease activity of ttMutS2 would be required for complete suppression of homologous recombination.…”

“…Plasmids pET-11a/ttmutS2, pET-11a/N60-ttmutS2, and pET-15b/ttSmr 621 , derived from pET-11a and pET-15b (Novagen), contain the complete, 3Ј-terminal region-deleted, and 5Ј-terminal region-deleted ttmutS2 genes, respectively, under control of a T7 promoter (17). ttMutS2, N60-ttMutS2, ttSmr 621 , and T. thermophilus MutS1 (ttMutS1) were overexpressed and purified as described previously (17,22). A DNA fragment expressing the ttSmr 663 domain was generated by PCR using the T. thermophilus HB8 genomic DNA as a template.…”

“…Gel Shift Assay-The assay was performed as described previously (17). The 32 P-labeled homoduplex and Holliday junction were incubated with 0 -1000 nM ttMutS2 or N60-ttMutS2 in 50 mM Tris-HCl, 100 mM KCl, 1 mM dithiothreitol, 0 or 5 mM ADP, and 0.1 mg/ml bovine serum albumin (BSA, Takara), pH 7.5, for 30 min at 37°C.…”

“…Nuclease Assay Using Plasmid DNA-The assay was performed as described previously (17). The 5 ng/l pUC19 (Takara) or pUC(AT) (New England Biolabs) plasmid DNA was incubated with or without freshly prepared ttMutS2 or bovine DNase I (Takara) in 50 mM TrisHCl, pH 7.5, 100 mM KCl, 5 mM MgCl 2 , 0.1 mg/ml BSA, and 1 mM dithiothreitol at 37°C.…”

“…Previously, we found that Thermus thermophilus MutS2 (ttMutS2) possesses nicking endonuclease activity (16), and this activity is confined to the C-terminal domain, which does not exist in the other MutS homologues (17). This C-terminal domain is called the small MutS-related (Smr) domain.…”