Binding of MutS protein to oligonucleotides containing a methylated or an ethylated guanine residue, and correlation with mutation frequency.
The MutS-based mismatch repair (MMR) system has been conserved from prokaryotes to humans, and plays important roles in maintaining the high fidelity of genomic DNA. MutS protein recognizes several different types of modified base pairs, including methylated guanine-containing base pairs. Here, we looked at the relationship between recognition and the effects of methylating versus ethylating agents on mutagenesis, using a MutS-deficient strain of E. coli. We find that while methylating agents induce mutations more effectively in a MutS-deficient strain than in wild-type, this genetic background does not affect mutagenicity by ethylating agents. Thus, the role of E. coli MMR with methylation-induced mutagenesis appears to be greater than ethylation-induced mutagenesis. To further understand this difference an early step of repair was examined with these alkylating agents. A comparison of binding affinities of MutS with O(6)-alkylated guanine base paired with thymine, which could lead to transition mutations, versus cytosine which could not, was tested. Moreover, we compared binding of MutS to oligoduplexes containing different base pairs; namely, O(6)-MeG:T, O(6)-MeG:C, O(6)-EtG:T, O(6)-EtG:C, G:T and G:C. Dissociation constants (K(d)), which reflect the strength of binding, followed the order G:T->O(6)-MeG:T->O(6)-EtG:T-=O(6)-EtG:C-> or =O(6)-MeG:C->G:C. These results suggest that a thymine base paired with O(6)-methyl guanine is specifically recognized by MutS and therefore should be removed more efficiently than a thymine opposite O(6)-ethylated guanine. Taken together, the data suggest that in E. coli, the MMR system plays a more significant role in repair of methylation-induced lesions than those caused by ethylation.
Taira
Kentaro
K
Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700-8530, Japan.
Nakamura
Shintaro
S
Nakano
Khota
K
Maehara
Daisuke
D
Okamoto
Keinosuke
K
Arimoto
Sakae
S
Loakes
David
D
Worth
Leroy
L
Schaaper
Roel M
RM
Seio
Kohji
K
Sekine
Mitsuo
M
Negishi
Kazuo
K
Negishi
Tomoe
T
D000477:Alkylating Agents / Q000494:pharmacology; D000478:Alkylation; D001483:Base Sequence; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005787:Gene Frequency; D006147:Guanine / Q000378:metabolism; D008969:Molecular Sequence Data; D051719:MutS DNA Mismatch-Binding Protein / Q000378:metabolism*; D009154:Mutation; D009841:Oligonucleotides / Q000378:metabolism*
D000477:Alkylating Agents; D029968:Escherichia coli Proteins; D009841:Oligonucleotides; D006147:Guanine; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli