The long term goal in the laboratory is to understand how DNA mismatch repair proteins and restriction -modification enzymes achieve their specificity.
Many types of restriction enzymes cleave DNA away from their recognition site. Using type III restriction enzyme, EcoP15I, that cleaves DNA 25 - 27 bp away from its recognition site, we provide evidence to show that intact recognition site on cleaved DNA sequesters the restriction enzyme and decreases the effective concentration of the enzyme. EcoP15I restriction enzyme is shown here to perform only single round of DNA cleavage. More importantly, we show that an exonuclease activity is essential for EcoP15I restriction enzyme to perform multiple rounds of DNA cleavage, which might hold true for all restriction enzymes cleaving DNA sufficiently away from recognition site. Our results highlight the importance of functional cooperation in the modulation of enzyme activity. We are currently investigating the details of how these enzymes recognize, cleave and modify DNA , both from a biochemical and structural standpoint.
With the availability of large quantities of N6-adenine methyltransferases (EcoP15I, EcoPI and KpnI) and a C5-cytosine methyltransferase (HhaI) we are interested in finding out how these enzymes recognize different sequences and carry out entirely different kinds of methylation reactions. Using steady-state kinetics and isotope-partioning experiments, the kinetic mechanisms of these methylases will be determined. We propose a detailed study of these enzymes using a variety of biochemical, biophysical and genetic approaches. These studies are thus aimed at understanding the precise interactions of the protein with DNA and to unravel the features of the enzyme action. It is hoped that these investigations would provide novel information on important molecular interactions.
Haemophilus influenzae is a wide spread human pathogen, responsible for several primary and secondary infections leading to a variety of diseased conditions. Recent genetic studies have shown that the DNA mismatch repair genes of H. influenzae have a major role to play in its pathogenecity mediated through a phenomenon called phase variation. The present study is aimed at the biochemical characterization of several mismatch repair proteins and in understanding protein-protein interactions among the mismatch repair proteins of Haemophilus influenzae.
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