Date of Award

1-1-2013

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Biomedical Sciences

Content Description

1 online resource (viii, 120 pages) : illustrations (some color)

Dissertation/Thesis Chair

Janice D Pata

Committee Members

Joachim Jaeger, Nilesh Banavali, Joseph Wade, Richard Cunningham

Keywords

PCNA, Polymerase fidelity, Pre-steady state, Translesional synthesis, X-ray crystallography, Y-family DNA polymerase, DNA polymerases, DNA replication

Subject Categories

Biochemistry | Biology | Biophysics

Abstract

Translesional synthesis (TLS) by specialized Y-family DNA polymerases is a DNA damage tolerance pathway to bypass DNA lesions that have not been repaired by other DNA repair mechanisms. Despite their valuable lesion bypass ability, the Y-family DNA polymerases display a much lower fidelity upon replicating undamaged DNA. Their activity therefore needs to be well regulated in the cell. In our first project, we examined how an archaeal Y-family DNA polymerase, Dpo4, makes single-base deletions during replication. Dpo4 belongs to the DinB subfamily which is known to make single-base deletions. Although previous studies suggested that it uses a dNTP-stabilized misalignment mechanism, our biochemical and structural data showed that Dpo4 uses a template-slippage mechanism to make single-base deletions. This is similar to the mechanism used by Dbh, a related Y-family DNA polymerases that we studied before. Our second project is concerned with the effects of the sliding clamp PCNA on the overall activity of Dbh. We found PCNA can moderately increase the polymerase activity and processivity of Dbh. An interesting finding is that PCNA interaction increases the fidelity of Dbh on a single-base deletion hot spot sequence by promoting insertion of the correct base. We also studied how different duplex lengths of linear primer-template DNA affect PCNA stimulation of Dbh activity. The two projects together provide us a better understanding of the mechanism and regulation of Y-family DNA polymerases.

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