Date of Award

1988

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Veterinary Medical Sciences - Pathobiological Sciences

First Advisor

H. Douglas Braymer

Abstract

The McrB restriction system in Escherichia coli K-12 is responsible for sequence-specific recognition and inactivation of DNA containing 5-methylcytosine. A derivative of plasmid pUC8 with a 5.5-kilobase pair BglII-EcoRI restriction fragment from the E. coli K-12 chromosome, imparted the wild-type phenotype to the McrB$\sp-$ strain K802. The limits of the McrB region within this DNA fragment were defined by deleting portions of the 5.5-kb insert and assaying for McrB restriction of M.AluI-methylated DNA. Analyses of polypeptides encoded by the McrB region using a maxicell strain revealed that at least three proteins, having molecular weights of approximately 51,000, 39,000, and 33,000, are produced. The 51-kDa protein, encoded by the mcrB gene, and the 39-kDa protein, encoded by the mcrC gene, together are required for McrB restriction activity in E. coli K-12. Transcription initiation assays and DNA sequence data indicated that transcription of the mcrB gene starts 710 base pairs beyond the termination codon of the hsdS gene, and proceeds in the same direction as transcription of the hsdR, hsdM, and hsdS genes of the EcoK, type I restriction system. The mcrC gene is located adjacent to the end of the mcrB gene encoding the carboxy terminus of its protein product. The nucleotide sequence confirmed the existence of two open reading frames corresponding to the 51-kDa and 39-kDa polypeptides, and also revealed a single nucleotide overlap between the termination codon of the mcrB gene and the proposed initiation codon of the mcrC gene. Analyses of plasmid-encoded proteins from several plasmid constructions containing frame-shift mutations brought about by a four nucleotide insertion in the McrB region, along with the DNA sequence data, provided evidence for a second start site for translation within the mcrB gene. Translation initiating at this second site, located 481 nucleotides downstream from the initiation codon for the 51-kDa protein, and continuing in the same reading frame is consistent with the production of the 33-kDa protein. The overlapping 33-kDa protein may possibly play a regulatory role in McrB-directed restriction. Another aspect of the McrB phenotype is the restriction of 5-hydroxymethylcytosine (HMC)-containing DNA from T-even phages in which the HMC residues are non-glucosylated. While the 39-kDa product of the mcrC gene is required to act together with the 51-kDa product of the mcrB gene to cause restriction of 5-methylcytosine DNA, this protein was shown to be unnecessary for restriction of bacteriophage T4 containing unmodified HMC residues.

Pages

109

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