Date of Award

1993

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Eric C. Achberger

Abstract

The McrBC (Modified Cytosine Restriction) restriction system has the ability to restrict DNA containing 5-hydroxymethylcytosine, N$\sp4$-methylcytosine, and 5-methylcytosine at specific sequences. The mcrB gene produces two gene products. The complete mcrB open reading frame produces a 53-kDa protein (McrB$\sb{\rm L}$) and a 35-kDa protein (McrB$\sb{\rm S}).$ The smaller McrB polypeptide is produced from an inframe, internal translational start in the mcrB gene. The mcrC gene produces a single 38-kDa protein. Evidence was presented that McrB$\sb{\rm S}$ regulates the activity of McrBC. When McrB$\sb{\rm S}$ was overproduced in a McrBC$\sp+$ background, there was dramatic loss of restriction. Underproduction of this protein using antisense RNA caused variable restriction and triggered the SOS response indicating extensive DNA damage. Based on experimental results, McrB$\sb{\rm S}$ was found to have distinct interactions with McrC and McrB$\sb{\rm L}$. A unique assay, termed the restriction rescue assay, was used to examine McrB$\sb{\rm S}$-McrC binding. Truncated versions of McrB$\sb{\rm S}$ were used in the McrB$\sp*$ assay to provide evidence that McrB$\sb{\rm S}$ and McrB$\sb{\rm L}$ bind. In some cases, elevated levels of McrB$\sp*$ activity, hyper-restriction, observed in the presence of McrB$\sb{\rm S}$ peptides was accompanied by induction of SOS, slow growth and cell death. Based on findings, we propose that the active restriction complex minimally consists of a McrB$\sb{\rm L}$ homodimer. McrB$\sb{\rm S}$ regulates restriction by binding McrB$\sb{\rm L}$ to form an inactive McrB$\sb{\rm L}$-McrB$\sb{\rm S}$ heterodimer. McrC associates with the active McrB$\sb{\rm L}$ homodimer to form the McrBC endonuclease and with McrB$\sb{\rm S}$ containing inactive complexes. Protein-DNA binding studies and in vitro cleavage studies allowed us to define specific binding site, cofactor, and subunit requirements for this restriction system. Results of these assays suggest that no subunit alone can bind specifically to DNA containing methylated PvuII sites. Target DNA fragments containing methylated PvuII sites were specifically bound when all three McrBC peptides were present whether these linear DNA fragments contained three, two, or one methylated PvuII sites.

Pages

117

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