Domain organization and functional analysis of Thermus thermophilus MutS protein [published erratum appears in Nucleic Acids Res 1998 Oct 15;26(20):following 4789]

Abstract

MutS protein binds to DNA and specifically recognizes mismatched or small looped out heteroduplex DNA. In order to elucidate its structure-function relationships, the domain structure of Thermus thermophilus MutS protein was studied by performing denaturation experiments and limited proteolysis. The former suggested that T. thermophilus MutS consists of at least three domains with estimated stabilities of 12.3, 22.9 and 30.7 kcal/mol and the latter revealed that it consists of four domains: A1 (N-terminus to residue 130), A2 (131-274), B (275-570) and C (571 to C-terminus). A gel retardation assay indicated that T.thermophilus MutS interacts non-specifically with double-stranded (ds), but not single-stranded DNA. Among the proteolytic fragments, the B domain bound to dsDNA. On the basis of these results we have proposed the domain organization of T. thermophilus MutS and putative roles of these domains.

Substoichiometric shifting in the plant mitochondrial genome is influenced by a gene homologous to MutS

“…2 A). This doublet was shown to be essential for mismatch recognition and specific DNA binding activity (33,34). We were unable to detect three other conserved domains characteristic of MutS.…”

DNA Binding and Protein-Protein Interaction Sites in MutS, a Mismatched DNA Recognition Protein from Thermus thermophilus HB8

“…With T. aquaticus MutS, a similar apparent molecular mass of 280 kDa was reported by size-exclusion chromatography (16,31); however, by other physicochemical methods, the molecular mass was estimated to be about 180 kDa, which corresponds to a dimer (31). Recently, it has been reported that E. coli MutS exists as mixture of dimers and higher order forms (17). The N-terminal and central domains were constructed as His-tagged proteins, and the resultant proteins were designated as MutS-A and MutS-B, respectively.…”

“…Domain-As we have already shown, MutS protein can be divided into at least three domains based on the results of limited proteolysis and the denaturation (17). In order to analyze the functions of each domain in more detail, truncated proteins that correspond to the proteolytic fragments were designed (Fig.…”

“…On the other hand, a phenylalanine residue in the N-terminal region of Thermus aquaticus MutS was found to be important for heteroduplex DNA binding (16). We have reported that the central region of Thermus thermophilus HB8 MutS binds homoduplex DNA (17). Recently, the N-terminal end and central regions of E. coli MutS were demonstrated to be required for binding of mismatched DNA (18).…”

“…T. thermophilus MutS protein is stable from pH 1.5 to 12 at 25°C and at a neutral pH up to 80°C (19). Results from protein denaturation and limited proteolysis experiments suggest that T. thermophilus MutS can be divided into three domains as follows: A (N terminus to residue 274), B (residues 275-570), and C (residue 571 to C terminus) (17). We have previously shown that the B domain interacts with double-stranded DNA (dsDNA); however, the assay system used in that study employed a mixture of all three domains.…”