Date of Defense

Fall 12-7-2000

Department

Biological Sciences

First Advisor

Karim Essani, Biological Sciences

Second Advisor

Fred L. Homa, Pharmacia Corporation

Third Advisor

Darrell R. Thomsen, Pharmacia Corporation

Abstract

The herpes simplex virus DNA polymerase gene, which plays an essential role in DNA replication, shares extensive amino acid homology with other alpha DNA polymerases. The sequences of the alpha DNA polymerases contain seven highly conserved regions. These regions are predicted to represent functional catalytic domains of the enzyme and help to accomplish basic steps for the DNA polymerization reaction. Research suggests that region III, which contains amino acid 823, is directly involved in substrate and drug recognition, deoxynucleoside triphosphate (dNTP) binding, and pyrophosphate (PPi) hydrolysis. Drug resistance is a possibility that all antiviral drugs must face. Studies of HSV drug resistant mutants have demonstrated that a large percentage of the mutations that confer resistance to the most commonly used HSV antivirals, including acyclovir, map to conserved region III. The focus of this study was to investigate what changes at amino acid 823 within conserved region III of the HSV polymerase would be tolerated and how those changes affect activity of the polymerase. To accomplish this, single amino acid changes were made at position 823 using site-directed mutagenesis through PCR techniques. This was then followed by analysis of the affects these changes had on polymerase production and activity. Sequencing of the recombinant genes indicated that six clones of the HSV-1 polymerase contained the correct mutation at amino acid 823. Polymerase activity analysis revealed that each of these genes produced polymerase of the same molecular weight as wild type virus. Through in vitro transcription/translation assays, each of the mutated polymerase genes was found to actively produce protein in varying amounts. These results will help to determine which HSV-1 mutations at amino acid 823 are tolerated and whether those changes make it behave like wild type or confer drug resistance in further studies of currently available herpesvirus drugs that target polymerase activity.

Access Setting

Honors Thesis-Campus Only

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