Date of Award

1-1-2012

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Biomedical Sciences

Content Description

1 online resource (xvii, 266 pages) : illustrations (some color)

Dissertation/Thesis Chair

Joachim Jaeger

Committee Members

Janice Pata, Marlene Belfort, Nilesh Banavali, Richard Cunningham

Keywords

base excision repair, DNA polymerase beta, DNA repair, human cancer, DNA polymerases, Mutation (Biology), Cancer cells

Subject Categories

Biochemistry | Molecular Biology

Abstract

DNA polymerases are essential for genome replication and DNA repair in all living organisms. Precise DNA replication is critical for the preservation of genomic stability. Any insult, endogenous/exogenous, to cellular DNA requires properly functioning repair polymerases. In eukaryotes, DNA polymerase beta, a small enzyme (39 kDa), plays an important role in DNA repair during the base excision repair pathway. Pol beta catalyzes the incorporation of nucleotides in small stretches (1-6 nucleotides) of damaged double-stranded DNA. Should gap-filling synthesis by pol beta be compromised, mutations in genomic DNA accumulate, which are frequently linked to human diseases, including cancers. For this reason, pol beta mutants have been extensively studied, particularly in terms of nucleotide incorporation kinetics. Interestingly, the numerous kinetics studies of these mutator mutants have revealed a link between residues distant from the active site and an increased rate of nucleotide misincorporation. Our experiments focus on mutator mutants located in the 8 kDa-N-terminal region (K72E), the hydrophobic hinge, linking the fingers and the palm domain (I260Q), the fingers domain (Y265H, H285D, E295K), and a very flexible loop in the palm domain (P242R, D246V, E249K). We augment biochemical and kinetic studies with structural data in order to further understand the importance of these distant residues and ultimately, to understand the mechanism by which these mutator mutants misincorporate nucleotides at the molecular level.

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