Date of Award

1-1-2014

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Biomedical Sciences

Content Description

1 online resource (xiii, 196 pages) : color illustrations

Dissertation/Thesis Chair

Janice D Pata

Committee Members

Joachim Jaeger, Marlene Belfort, Haixin Sui, Graham Walker

Keywords

C-family, DNA polymerase, PolC, replication, translesion synthesis, Y-family, DNA polymerases, DNA repair, DNA replication, Staphylococcus aureus

Subject Categories

Biochemistry

Abstract

Since the discovery of the DNA polymerase by Arthur Kornberg nearly 60 years ago, there have been great advances in understanding the involvement of polymerases in replication and repair. Years of genetic, biochemical and structural studies have lead to the classification of DNA-dependent DNA polymerases into six families: A, B, C, D, X and Y. In this work, I have focussed on two different families, C and Y. Hence this work is divided into two parts. Part one discusses the studies on Y-family polymerases. All Y-family polymerases are involved in replicating past DNA lesions. The ability to tolerate unnatural nucleotides also makes Y-family enzymes extremely error-prone. Here I specifically focus on Y-family members belonging to the DinB category. DinB homologs are known to generate frameshift mutations and single base deletions at high frequencies, particularly on repetitive undamaged DNA sequences. I have determined the mechanism that is used by pol kappa (κ), the DinB homolog in humans, to generate single-base deletions on a homopolymeric run. We observe that DinB polymerases realign their susbtrate to convert deletions to base-substitution mutations. The importance of this observation is discussed. To better understand which element of Y-family polymerase architecture determines the error-generation specificity of these polymerases, I have used two closely related DinB homologues in archaea (Dbh and Dpo4) to generate chimeric constructs. Our biochemical and crystallographic evidence indicates that a linker connecting the polymerase domain to the C-terminal little finger/polymerase-associated domain (LF/PAD) in both these polymerases is crucial for determining conformation and mutation signature. The altered conformation specifically influenced the rate of correct nucleotide incorporation, thereby determining polymerase fidelity. The relevance of this result is also discussed. The second part of this thesis is aimed at filling the distinct gap that exists in our understanding of the kinetic pathway of bacterial replicative DNA polymerases. I have addressed this by using detailed pre-steady state and steady state assays to determine the kinetic parameters for the DNA polymerization pathway followed by the bacterial C-family replicative polymerase, PolC from the Gram positive pathogen Staphylococcus aureus. We find PolC exhibiting several unusual characteristics, the most interesting one being slow pyrophosphate release. Our studies lead to the surprising observation that PolC can overcome its slow PPi release, a critically limiting step, when the next incoming nucleotide is present. This novel observation and its implications are further discussed.

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