Date of Award

2005

Degree Name

Doctor of Philosophy

College

Joan C. Edwards School of Medicine

Type of Degree

Ph.D.

Document Type

Dissertation

First Advisor

Terry W. Fenger

Second Advisor

Elizabeth C. Bryda

Third Advisor

Donald A. Primerano

Abstract

Polymerase stop assays, used to quantify DNA damage, assume single lesions are sufficient to block thermostable DNA polymerase progression. To explore this assumption, 90 base oligonucleotides containing normal or modified DNA bases were amplified using real-time PCR. Data implied that the PCR efficiency was influenced to differing degrees depending on which base lesion was present on the input oligonucleotide; specifically, while reactions with templates containing a single 8-oxo-7,8-dihydro-2í-deoxyguanosine (8-oxodG) were not noticeably altered, the presence of a single 8-oxo-7,8-dihydro-2í-deoxyadenosine, an abasic site, or a cis-syn thymidine dimer (TT dimer) dramatically delayed amplification. In addition, the presence of two tandem 8-oxodGs substantially hindered amplification when compared with two 8-oxodGs separated by 13 bases which indicated that the position of lesions also influenced the PCR. To quantify variations in amplification, novel mathematical formulae were developed which report differences in exponential amplification as rates of damage bypass. These treatments assume each template in the PCR is damaged to the same degree. Quantification of damage to cellular DNA, which is a mixture of damaged and undamaged template, required further refinement of real-time PCR mathematics; differences in amplification were defined in terms of damage probability (lesion frequency) rather than lesion bypass rate. The validity of these formulae was determined using DNA samples quantified previously using current polymerase stop methods. In addition to impacting reaction efficiency, DNA base modifications decreased reaction fidelity. In reactions with templates containing 8-oxodGs, both the normal Watson/Crick association with dCMP as well as the incorporation of dAMP occurred at the lesion site. Despite similar structural characteristics, the existence of 8-oxodA resulted in a pronounced n-1 deletion in addition to the normal association with dTMP. Sequence data from abasic and TT dimer modifications were inconclusive but suggested the presence of multiple nucleotide incorporation events opposite the modifications. The present work enabled the adaptation of real-time PCR for DNA damage quantification, identified DNA base lesions as potential PCR mutagens, and provides the basis for further refinement of polymerase stop assays as research and clinical tools to monitor DNA damage and repair.

Subject(s)

DNA damage.

DNA polymerases.

Polymerase chain reaction.