Functional characterization of the DNA mismatch binding protein MutS from Haemophilus influenzae.
This investigation demonstrates DNA mismatch repair activity in Haemophilus influenzae cell free extracts. The mutS gene as well as purified protein of H. influenzae restored repair activity in complementation assays performed with mutS deficient Escherichia coli strain. The difference in affinity for GT and AC mismatched bases by H. influenzae MutS was reflected in the efficiency with which these DNA heteroduplexes were repaired in vitro, with GT being repaired well and AC the least. Unlike E. coli MutS, the H. influenzae homolog failed to give protein-DNA complex with homoduplex DNA. Interestingly, MutS was found to bind single-stranded DNA but with lesser affinity as compared to heteroduplex DNA. Apart from the nucleotide- and DNA-mediated conformational transitions, as monitored by circular dichroism and limited proteolysis, our data suggest a functional role when H. influenzae MutS encounters single-stranded DNA during exonucleolytic step of DNA repair process. We propose that, conformational changes in H. influenzae MutS not only modulate mismatch recognition but also trigger some of the down stream processes involved in the DNA mismatch repair process.
Joseph
Nimesh
N
Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India.
Duppatla
Viswanadham
V
Rao
Desirazu N
DN
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D020137:Base Pair Mismatch; D004260:DNA Repair / Q000502:physiology*; D004926:Escherichia coli / Q000235:genetics; D005816:Genetic Complementation Test; D006193:Haemophilus influenzae / Q000737:chemistry*; D051719:MutS DNA Mismatch-Binding Protein / Q000172:deficiency / Q000502:physiology*; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects
D009692:Nucleic Acid Heteroduplexes; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D051719:MutS DNA Mismatch-Binding Protein