Date of Award

1998

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Eric C. Achberger

Abstract

The McrBC system of Escherichia coli K12 restricts DNA containing modified cytosines within specific sites. The modified cytosines recognized may be 5-hydroxymethylcytosine, N$\sp4$-methylcytosine or 5-methylcytosine. In the mcrBC operon, the mcrB gene is expressed to produce the McrB$\sb{\rm L}$ protein (51 kDa) and the McrB$\sb{\rm S}$ protein (33kDa), which is translated from an inframe internal translation start site and lacks the N-terminal 161 amino acids of the long product. The McrC protein is a 39kDa protein and is required for the cleavage of most methylated substrates. Using a MalE-McrB$\sb{\rm L}$ fusion protein, the endonucleolytic McrB$\sb{\rm L}$ subunit was shown to form a stable dimer. The interactions of the MalE-McrB$\sb{\rm L}$ fusion protein with McrB$\sb{\rm S}$ and McrB$\sb{\rm L}$ subunits was also demonstrated in a western blot analysis. A MalE-McrC fusion protein was shown to bind McrB$\sb{\rm L}$ and McrB$\sb{\rm S}$ providing additional biochemical evidence for the interactions among the various subunits of the McrBC system. The sites of DNA binding by the McrBC subunits were mapped using the DNase I footprinting technique. The DNA binding subunit of the McrBC system was demonstrated to be the McrB$\sb{\rm L}$ protein. The McrC subunit was not essential for the specificity of the McrBC endonuclease. The role of the subunit composition on DNA binding was also measured using DNase I footprinting technique. Elevated levels of MalE-McrC fusion protein were found to lower DNA binding by the McrB peptides. DNA binding studies for targets with two and three PvuII sites suggested cooperative binding of the McrB dimers at two adjacent methylation sites. A DNA target with a single methylation site was found to be bound by McrBC weakly. Primer extension analysis was used to examine the cleavage activity of the McrBC enzyme complex and a 65 amino acid fragment of the McrB$\sb{\rm S}$ subunit. The truncated form of McrB$\sb{\rm S}$ contained a portion of the GTP binding site, and was found to be a cofactor independent, non-specific endonuclease with nicking activity. The portion of the mcrB gene expressing the truncated protein was used to overexpress and analyze it as a His-tagged peptide. A unifying model describing the subunit structure and DNA binding properties is presented.

ISBN

9780591766615

Pages

95

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