Cruciform Extrusion in Methylation-State Variants of pBR322 Plasmid

Author

Lucas Morgan McElwain

Date of Award

2007

Document Type

Undergraduate Thesis

Department

Chemistry and Biochemistry

First Advisor

Randy Wadkins

Relational Format

Dissertation/Thesis

Abstract

The presence of DNA secondary structures in genomic DNA has been observed in both prokaryotes and eukaryotes. Specifically, sections of ssDNA may melt out and form cruciform/hairpin structures within palindromic sequences or areas of high purine content. Negative supercoiling of the DNA is necessary for energetic favorability of such an event. These noncanonical structures may serve as molecular switches, providing additional regulation of DNA transactions such as transcription and perhaps replication and recombination. Secondary structures associated with regulatory regions are of particular interest as potential sites for the binding of small molecules, which could alter the expression of genes. The effect of system-wide DNA base methylation on extrusion of secondary structures is one aspect of primary-secondary-tertiary structure interplay that has not been examined. Our study first characterized the gross qualitative effect of methylation of plasmid DNA on the formation of cruciform structures. The plasmid pBR322 was transformed into three different strains of E. coli: K12 with functional deoxycytosine and deoxyadenine methyltransferases, BL21 strain (dcm ), and K12 ER2925 with no methyltransferase activity (dam Idem). The resulting purified plasmids were then digested with mung bean nuclease (MBN); a ssDNA-specific nuclease; and then by a site-specific nuclease. The digest reaction mixtures were analyzed by gel electrophoresis for fragments characteristic of cleavage by MBN. The three strains seemed to show no difference in the presence of at least one prominent ssDNA site. Topoisomers of each plasmid were then created by the relaxation of negative supercoils in the presence of DNA intercalator and separated through gel electrophoresis. The three methylation-state variants seemed to exhibit similar structural responses. These data indicate that methylation of respective nucleotide bases does not seem to influence cruciform extrusion, through either changes in local or global conformations.

Recommended Citation

McElwain, Lucas Morgan, "Cruciform Extrusion in Methylation-State Variants of pBR322 Plasmid" (2007). Honors Theses. 2236.
https://egrove.olemiss.edu/hon_thesis/2236

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