The Effects of DNA Binding Proteins on Dam Processivity
Authors:
Catherine Nguyen, Jennifer PerdomoMentor:
Stacey Peterson, Associate Professor, Mount St. Mary's CollegeEscherichia coli DNA adenine methyltransferase (Dam) is an enzyme which has an important role in DNA replication, gene expression, and mismatch repair. Dam specifically methylates adenine within GATC sequences in DNA. Dam has been shown to be processive, staying bound to DNA until it has methylated multiple GATC sites. In vivo, Dam is responsible for methylating approximately 20,000 GATC sites on the bacterial chromosome. Other proteins on the chromosome may either enhance or interrupt Dam’s processivity. Possible interruptions could cause Dam to function insufficiently, leading to problems such as mutations in newly replicated DNA, or differences in expression of genes. In order to test the effect of DNA-binding proteins on Dam processivity, we used a relatively small linear fragment of DNA with two GATC sites, separated by Lrp binding sites. Lrp is a nucleoid-associated protein that binds DNA specifically or nonspecifically and is found throughout the chromosome, therefore likely encounters Dam. Furthermore, Lrp bends DNA which may help Dam methylate more efficiently by bringing consecutive GATC sites closer together. We perfomed in vitro processivity assays in the presence and absence (control) of LRP. To identify the methylation of Dam we added the restriction enzyme DPN II which cuts DNA at non-methylated GATC sites. Our findings thus far show that Dam is less processive and its methylation rate is slower in the presence of Lrp. We will continue to perform these experiments to confirm the effect is reproducible. In the future, we plan to use different DNA substrates and other DNA binding proteins to analyze their effect on Dam processivity.