GSBS Dissertations and Theses

Approval Date

11-1-1991

Document Type

Doctoral Dissertation

Department

Graduate School of Biomedical Sciences, Pharmacology

Subjects

Escherichia coli; DNA Repair; Academic Dissertations; Dissertations, UMMS

Abstract

The Dam-dependent mismatch repair system of Escherichia coli is part of a large network of DNA surveillance and error avoidance systems that identify and repair DNA damage. In this thesis, I have investigated the Dam-dependent mismatch repair system of E. coli and its role in the recognition and repair of DNA substrate molecules containing small insertion/deletion heterologies. This investigation was divided into two parts: the first part utilized genetic techniques to evaluate the specificity of repair and the second part utilized biochemical approaches into the recognition of insertion/deletion heterologies.

I have developed a sensitive in vivo transformation system to rapidly evaluate the repair of small insertion/deletion heterologies by Dam-dependent mismatch repair. Heteroduplexes were constructed, for each state of methylation of d(GATC) sequences, by annealing single strand DNA to the linearized complementary strand of duplex DNA. The unmethylated single strand DNA was isolated from f1 phage (R408) propagated on a strain of E. coli containing the dam-16 allele (Chapter 2) to eliminate the possibility of residual Dam-methylation of d(GATC) sequences. Tranformation of E. coli indicator strains with heteroduplexes containing 1, 2, 3, 4 and 5 base insertion/deletion heterologies were scored for repair based on colony color. The results of these experiments show that the Dam-dependent mismatch repair system can recognize and repair 1, 2 and 3 base heterologies as well as repairing G/T mispairs (Chapters 3 and 4). The repair of 4 base heterologies was marginal, while no repair was observed with 5 base heterologies (Chapters 3 and 4). Repair of the 1, 2, 3 and 4 base heterologies proceeded in a Dam-dependent process that required the gene products of mutL, mutS, and

I have demonstrated that MutS protein from both Salmonella typhimurium and E. coli can recognize and bind in vitro to the same 1, 2, 3 and 4 base heterologies used for the genetic studies above (Chapters 4 and 5). In fact, MutS protein binds to 1, 2 and 3 base heterologies with greater affinity than it binds to a G/T mismatch. The in vitro observation that MutS does not bind to 5 base heterologies is consistent with the in vivo observation that 5 base heterologies are not subject to repair. I have also shown that MutS protein specifically binds to 1, 2 and 3 base heterologies since MutS protects about 25 base pairs of DNA flanking the site of the heterology from DNaseI digestion.

The results of the genetic and biochemical experiments described in this thesis (and summarized above) serve to re-emphasize the importance of the role that methyl-directed mismatch repair plays in mutation avoidance, and hence in the preservation of genetic integrity.

Comments

Some images did not scan well. Please consult original document. In the abstract, the second paragraph ends in mid-sentence.

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Copyright is held by the author, with all rights reserved.

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