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Structural Mechanisms of Accurate and Mutagenic DNA Replication

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Date
2012
Author
Wang, Weina
Advisor
Beese, Lorena S
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Abstract

High-fidelity DNA polymerases achieve remarkable accuracy by exhibiting exquisite nucleotide substrate selection mechanisms. DNA polymerases not only discriminate highly against base-pair mismatches, but also show a high degree of selectivity for the correct sugar moiety deoxyribonucleotide over ribo- and dideoxy-nucleotides. Although DNA polymerase is highly accurate, some base-pair mismatches are still incorporated at low frequency, leading to spontaneous mutagenesis. Mechanisms for both accurate and mutagenic DNA replication have been subjected to intense solution studies and speculations over half a decade. However, structural understanding of both processes are still very limited due to the lack of crystal structures of DNA polymerase bound with incorrect nucleotide substrates especially at the insertion step prior to chemistry which accounts for the majority of the intrinsic specificity of the polymerase.

In this dissertation, X-ray crystallographic analyses were performed using a model system for studying replication fidelity, a thermostable strain of Bacillus DNA polymerase I large fragment (Bacillus fragment, BF) that catalyzes replication in crystals. A series of high-resolution crystal structures of BF polymerase variants and DNA duplex with mismatches, incorrect sugar substrates, and cognate base pairs bound at various fidelity filter sites on the polymerase surface were determined. By comparing structures of non-cognate base pairs with those of cognate base pairs captured under the same experimental condition, a unified picture of substrate selectivity by DNA polymerase for both mutagenic and accurate replication process has emerged.

Type
Dissertation
Department
Biochemistry
Subject
Biochemistry
Permalink
https://hdl.handle.net/10161/5607
Citation
Wang, Weina (2012). Structural Mechanisms of Accurate and Mutagenic DNA Replication. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/5607.
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