Deoxyguanosine triphosphate, a possible target for reactive oxygen species-induced mutagenesis Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/td96k555z

Descriptions

Attribute NameValues
Creator
Abstract or Summary
  • Intracellular dNTP pool sizes are highly asymmetric, with dGTP usually comprising 5 to 10% of the sum of the dNTP pools. The work presented in this dissertation addresses the question of whether the underrepresentation of dGTP is related to its potential to be oxidized by reactive oxygen species. 8-oxo-guanine is important in oxidative mutagenesis, and current evidence indicates that this lesion arises in DNA partly through oxidation of dGTP, followed by incorporation of 8-oxo-dGTP into DNA. The bacterial MutT protein and its mammalian homolog catalyze the hydrolysis of 8-oxo-dGTP to 8-oxo-dGMP in vitro. It is a widely accepted premise that the primary function of these enzymes is to remove 8-oxo-dGTP from the nucleotide pool of cells so that it cannot be used as a substrate for DNA synthesis. However, this model has been called into question by observations that some mutT strains of E. coli display a mutator phenotype when grown anaerobically, and by kinetic studies that showed 8-oxo-dGTP to be a poor DNA polymerase substrate. In this study, the dNTP pools of mammalian cells cultured in varying oxygen conditions were measured, with the expectation that the dGTP pool would expand under low oxygen conditions if it were a target for damage by reactive oxygen species. HeLa cells cultured in 2% 0₂ showed no change in the dGTP pool when compared to cells cultured in 20% 0₂; however, in V79 cells, the dGTP pool did expand in 2% 0₂. This result was not specific to the dGTP pool, as pools of dATP and dTTP also increased when V79 cells were cultured at 2% 0₂. These results suggest that there may be increased turnover of the dGTP pool when cells are cultured in high oxygen, but these experiments did not address the reason for this oxygen-dependent change. In order to determine whether 8-oxo-dGTP accumulates to levels that are sufficient to cause mutagenesis in cells, an analytical method for the measurement of 8-oxo-dGTP from cell extracts was developed. By use of this method, which involves reversed-phase high performance liquid chromatography coupled with electrochemical detection, no 8-oxo-dGTP was detected in mutT E. coli cells, even when they were cultured in the presence of H₂0₂. The estimated upper limit of 8-oxo-dGTP in these cells is about 240 molecules per cell, which corresponds to an intracellular concentration of approximately 0.34 μM. When 8-oxo-dGTP was added at this concentration to an in vitro DNA replication system in which replication errors could be scored as mutations, along with the four normal dNTPs at their estimated intracellular concentrations, there was no detectable effect on the frequency of mutation. Therefore, the presence of 8-oxo-dGTP at physiologically relevant concentrations does not appear to be significantly mutagenic. The results presented in this dissertation suggest that the mechanism by which the MutT enzyme counteracts mutagenesis should be reevaluated.
Resource Type
Date Available
Date Copyright
Date Issued
Degree Level
Degree Name
Degree Field
Degree Grantor
Commencement Year
Advisor
Non-Academic Affiliation
Subject
Rights Statement
Peer Reviewed
Language
Digitization Specifications
  • File scanned at 300 ppi (Monochrome) using Capture Perfect 3.0 on a Canon DR-9050C in PDF format. CVista PdfCompressor 4.0 was used for pdf compression and textual OCR.
Replaces
Additional Information
  • description.provenance : Approved for entry into archive by Patricia Black(patricia.black@oregonstate.edu) on 2012-07-13T18:00:50Z (GMT) No. of bitstreams: 1 TassottoMaryLynnBenka2003.pdf: 1163470 bytes, checksum: 8e97b82293cb8edecbeb5f9bb6ebddc9 (MD5)
  • description.provenance : Approved for entry into archive by Patricia Black(patricia.black@oregonstate.edu) on 2012-07-13T18:06:02Z (GMT) No. of bitstreams: 1 TassottoMaryLynnBenka2003.pdf: 1163470 bytes, checksum: 8e97b82293cb8edecbeb5f9bb6ebddc9 (MD5)
  • description.provenance : Submitted by Eric Hepler (ehscanner@gmail.com) on 2012-07-10T19:38:42Z No. of bitstreams: 1 TassottoMaryLynnBenka2003.pdf: 1163470 bytes, checksum: 8e97b82293cb8edecbeb5f9bb6ebddc9 (MD5)
  • description.provenance : Made available in DSpace on 2012-07-13T18:06:02Z (GMT). No. of bitstreams: 1 TassottoMaryLynnBenka2003.pdf: 1163470 bytes, checksum: 8e97b82293cb8edecbeb5f9bb6ebddc9 (MD5) Previous issue date: 2002-09-04

Relationships

In Administrative Set:
Last modified: 08/15/2017

Downloadable Content

Download PDF
Citations:

EndNote | Zotero | Mendeley

Items