PMID:12488096
| Citation |
Friedhoff, P, Thomas, E and Pingoud, A (2003) Tyr212: a key residue involved in strand discrimination by the DNA mismatch repair endonuclease MutH. J. Mol. Biol. 325:285-97 |
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| Abstract |
The molecular mechanism of how the dam-methylation status of the DNA is recognized during DNA mismatch repair by the strand discrimination endonuclease MutH is not known. A comparison of the crystal structure of MutH with those of co-crystal structures of several restriction endonucleases, together with a multiple sequence alignment of MutH and related proteins suggested that Phe94, Arg184 and Tyr212 could be involved in discrimination between a methylated or unmethylated adenine in the d(GATC) sequence. A mutational analysis revealed that the variants R184A and Y212S, but not F94A, were substantially reduced in their ability to complement a mismatch repair deficiency in a mutH(-) Escherichia coli strain. In vitro, R184A displayed a strongly reduced endonuclease activity, whereas the Y212S variant has almost completely lost its preference for cleaving the unmethylated strand at hemimethylated d(GATC) sites. Furthermore, the Y212 variant can cleave fully methlyated d(GATC) sites at a comparable rate to unmethylated d(GATC) sites. This demonstrates that Tyr212 is an important, if not the only amino acid residue in MutH for sensing the methylation status of the DNA. |
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| Keywords |
Amino Acid Sequence; Base Pair Mismatch; Binding Sites; Circular Dichroism; DNA Repair; DNA Repair Enzymes; DNA, Bacterial/metabolism; DNA-Binding Proteins/chemistry; DNA-Binding Proteins/genetics; DNA-Binding Proteins/metabolism; Endodeoxyribonucleases/chemistry; Endodeoxyribonucleases/genetics; Endodeoxyribonucleases/metabolism; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli Proteins; Genetic Complementation Test; Methylation; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Structure, Tertiary; Sequence Alignment; Tyrosine/metabolism |
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