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

Abstract

The mismatch repair system repairs mismatched base pairs, which are caused by either DNA replication errors, DNA damage, or genetic recombination. Mismatch repair begins with the recognition of mismatched base pairs in DNA by MutS. Protein denaturation and limited proteolysis experiments suggest that Thermus thermophilus MutS can be divided into three structural domains as follows: A (N-terminal domain), B (central domain), and C (C-terminal domain) (Tachiki, H., Kato, R., Masui, R., Hasegawa, K., Itakura, H., Fukuyama, K., and Kuramitsu, S. (1998) Nucleic Acids Res. 26, 4153-4159). To investigate the functions of each domain in detail, truncated genes corresponding to the domains were designed. The gene products were overproduced in Escherichia coli, purified, and assayed for various activities. The MutS-MutS protein interaction site was determined by size-exclusion chromatography to be located in the B domain. The B domain was also found to possess nonspecific double-stranded DNA-binding ability. The C domain, which contains a Walker's A-type nucleotide-binding motif, demonstrated ATPase activity and specific DNA recognition of mismatched base pairs. These ATPase and specific DNA binding activities were found to be dependent upon C domain dimerization.In living organisms, DNA damage often arises as a result of errors introduced by DNA replication, genetic recombination, and other processes (1). These DNA lesions can result in mutations, genetic diseases, and tumors. To remove such lesions, all organisms have developed DNA repair systems. The mismatch repair (MMR) 1 system is one of such repair systems and is conserved significantly throughout all organisms. In Escherichia coli, MutS, MutL, and MutH proteins are included in the MMR system (2). The pathogenic genes of human hereditary nonpolyposis colorectal cancer appear to share a high degree of homology (ϳ30%) with bacterial MutS and MutL (3, 4). Moreover, MutS homologues have also been isolated from plants (5).These observations suggest that the MMR system is essential for all living organisms from bacteria to eukaryotes. Recently, the three-dimensional structures of E. coli MutL and MutH have been reported, and the relationships between structures and functions have been addressed (6 -8).MutS plays a key role in the early processes of MMR, mediating mismatched base pair recognition. Eukaryotic MutS homologues have been found to bind to mismatched/looped out DNA (9 -12). It has also been reported that E. coli MutS binds to mismatched DNA as a dimer, forming an ␣-shaped loop structure (13). This finding suggests that this protein has two DNA-binding sites, one of which binds to mismatched DNA and the other to homoduplex DNA. There have been many studies exploring the DNA-binding region of MutS. A C-terminal mutant of Salmonella typhimurium MutS was found to have reduced affinity for heteroduplex DNA (14). Furthermore, the C-terminal region of hMSH2, a human MutS homologue, has been shown to be sufficient for binding to mismatches in DNA (15). On the other han...

DNA Mismatch Repair in Eukaryotes and Bacteria

“…Bacterial MutS forms a homodimer and recognizes mispaired bases and short insertion/deletion loops [9, 40, 41]. Eukaryotes possess several mismatch-recognizing MutS homologues: MSH2, MSH3, and MSH6.…”

Molecular Mechanisms of the Whole DNA Repair System: A Comparison of Bacterial and Eukaryotic Systems

“…First, T. thermophilus MutS exhibits a high affinity for mismatched heteroduplexes [138, 155], and the mismatch-MutS complex seems to be stabilized by MutL [152]. Second, similar ATP binding-dependent conformational changes have been observed in MutS homologues from T. thermophilus [156], E. coli [157, 158], and humans [159, 160].…”

Activity of Vsr endonucleases encoded by Neisseria gonorrhoeae FA1090 is influenced by MutL and MutS proteins

“…gonorrhoeae and E. coli MutL proteins and the high evolutionary conservation of MutL proteins. Such conservation of the N-terminal domain has been observed for MutL proteins from different bacterial species [ 31 ] and may indicate a universal mechanism of Vsr activity regulation by MutL protein that involves its N-terminal domain. Indeed, in the E. coli MutL protein, the N-terminal domain is responsible for physical interactions with V.EcoKDcm endonuclease [ 4 ].…”