Requirement for Phe36 for DNA binding and mismatch repair by Escherichia coli MutS protein.
The MutS family of DNA repair proteins recognizes base pair mismatches and insertion/deletion mismatches and targets them for repair in a strand-specific manner. Photocrosslinking and mutational studies previously identified a highly conserved Phe residue at the N-terminus of Thermus aquaticus MutS protein that is critical for mismatch recognition in vitro. Here, a mutant Escherichia coli MutS protein harboring a substitution of Ala for the corresponding Phe36 residue is assessed for proficiency in mismatch repair in vivo and DNA binding and ATP hydrolysis in vitro. The F36A protein is unable to restore mismatch repair proficiency to a mutS strain as judged by mutation to rifampicin or reversion of a specific point mutation in lacZ. The F36A protein is also severely deficient for binding to heteroduplexes containing an unpaired thymidine or a G:T mismatch although its intrinsic ATPase activity and subunit oligomerization are very similar to that of the wild-type MutS protein. Thus, the F36A mutation appears to confer a defect specific for recognition of insertion/deletion and base pair mismatches.
Yamamoto
A
A
Genetics and Biochemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-1810, USA.
Schofield
M J
MJ
Biswas
I
I
Hsieh
P
P
D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate / Q000378:metabolism; D000409:Alanine / Q000502:physiology; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D020137:Base Pair Mismatch; D004260:DNA Repair; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D029968:Escherichia coli Proteins; D006868:Hydrolysis; D008969:Molecular Sequence Data; D051719:MutS DNA Mismatch-Binding Protein; D009154:Mutation; D010649:Phenylalanine / Q000502:physiology*; D011485:Protein Binding
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D010649:Phenylalanine; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli; D000409:Alanine