Characterization of the DNA sequence specificity of the wildtype and hypermethylating mutant forms of the phages T2 and T4 DNA-Adenine methylase (dam) and the Eco dam methylase of the host bacterium, Escherichia coli

Author

Mark M. Doolittle

Date of Award

8-1987

Degree Type

Thesis

Degree Name

Master of Science

Major

Microbiology

Major Professor

Karl M. Sirotkin

Committee Members

Jeffrey Becker, William Riggsby, Gary Sayler

Abstract

The use of oligonucleotide nearest neighbor analysis or protection from restriction endonuclease cleavage assays has not proven to be rigorous enough to definitively determine the sequence specificity of the T₂ and T₄⁺ dam and dam^{h methylases. Therefore, a novel assay was developed to qualitatively and quantitatively determine the specificity of the phage dam methylases and the host methylase, Escherichia coli Eco dam. Plasmid (pBR322) DNA, whose sequence is known, was subjected to methylation in the presence of the radiolabelled methyl donor 3H-S-adenosylmethionine (3H-SAM) then purified using standard nucleic acid techniques. Plasmid restriction fragments, containing one or very few possible methylation sites were isolated from resolving polyacrylamide gels and analyzed by scintillation counting to determine the extent of methylation.}

It was established that the Eco dam enzyme only methylated the canonical site (GATC), while the T₂ and T₄ dam enzymes also methylated other GAPyN sites. More specifically, the T₂dam⁺ enzyme methylated certain GATG and GAGC sites (in addition to the canonical GATC site) more readily than the T₄dam⁺ enzyme. The degree to which noncanonical GAPyN sites were methylated by the T₂dam⁺ enzyme was shown to be affected by pH and concentration of s-adenosylmethionine (SAM). GAPyN sites, other than the canonical site (GATC) were methylated more readily in methylation reactions containing higher concentrations of SAM. The T and T₄dam^h enzymes differed from their wildtype counterparts in that they methylated GACC sites more readily at lower SAM concentrations. DNA substrate topology (supercoiling) was shown to alter site preference only slightly.

Recommended Citation

Doolittle, Mark M., "Characterization of the DNA sequence specificity of the wildtype and hypermethylating mutant forms of the phages T2 and T4 DNA-Adenine methylase (dam) and the Eco dam methylase of the host bacterium, Escherichia coli. " Master's Thesis, University of Tennessee, 1987.
https://trace.tennessee.edu/utk_gradthes/13453