Title |
In Vivo Analysis of the Specificity of DNA
Adenine Methyltransferase Mutants R116A and N126A |
Except for prepositions, only the First Letter of each word should be capitalized. Only scientific names of organisms (genus and species) should be in italics. Do not bold any part of the title. Do not add a period at the end of the title
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Author Block |
Ana C. Castillo, and Stacey N. Peterson, Ph.D.
Mount St. Mary's College, Los Angeles, CA.
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Abstract |
DNA adenine methyltransferase (Dam) is responsible for successful mismatch repair, replication timing, and the expression of several pathogenic genes in Escherichia coli (E. coli).
Dam methylates GATC sequences at the N6 position of the adenine. The three base pairs flanking the 5’ and 3’ ends of a GATC site have been shown to influence the methylation
efficiency of Dam for that site. Both in vitro and
in vivo studies have demonstrated that methylation of GATC sites with A/T rich flanking sequences are less efficiently methylated (“bad” sites) than those with G/C rich flanking
sequences (“good” sites). Our goal is to
determine how the structure of Dam contributes to this preference for certain GATC sites and what residues are involved. The E. coli Dam/DNA co-crystal structure reveals Dam residues that lie within close proximity to sequences flanking the GATC. The side chains of some of these residues are within hydrogen bonding distances to the phosphate backbone of the GATC flanking sequences. Two of these residues, R116 and N126, were selected for study and independently mutated to alanine, forming Dam mutants R116A and N126A. We hypothesize that disruption of the hydrogen bonds between the GATC flanking sequences and the amino acid side chains of R116 or N126 may will affect the methylation efficiency of Dam at “good” and “bad” GATC sites. To test this hypothesis, we transformed dam-minus E. coli cells with one of three plasmids. One plasmid contained the wildtype dam gene, one the R116A mutant dam, and one the N126A mutant dam, each under control of the arabinose promoter. The plasmids also contained two consecutive “good” and “bad” GATC sites for simple analysis of methylation preference. The transformed cells were grown with varying amounts of arabinose to monitor expression levels of Dam. The plasmids were purified and methylation of “good” and “bad” sites was analyzed using methylation-sensitive restriction enzymes. Our preliminary results reveal that both mutants show a difference in GATC site preference compared to the wildtype, suggesting the importance of these residues in specificity. Analysis of the R116A and N126A mutants will help in the characterization of the Dam protein and will illuminate an alternate route for the control of bacterial DNA methylation that is crucial for bacterial pathogenesis. |
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