Damerla, Rama Rao
(2011)
Mutagenic Potential of Telomeric Repeats and the Role of Werner Syndrome Helicase Protein in Facilitating Telomeric DNA Replication.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Chromosome termini form nucleoprotein structures called telomeres that consist of tandem repeats of TTAGGG DNA sequences (mammals) and telomeric proteins. Telomeres play a critical role in cell survival and genomic stability. Biochemical studies showed that the G-rich strand of telomeres can fold into secondary DNA structures called G-quadruplexes (G4-DNA), which are thought to impact telomere length regulation and telomeric DNA stability. G4 DNA structures are capable of interfering with DNA synthesis by blocking DNA polymerases in vitro and are proposed to hinder replication in vivo. We cloned telomeric repeats into reporter cassettes on shuttle vectors and replicated them in normal human somatic cells to determine if telomeric repeats induce mutations and deletions due to their ability to fold into G4 DNA structures. We demonstrated for the first time that G-rich telomeric repeats, in spite of their G4 DNA forming ability are stable upon replication in normal human cells. In contrast, ciliate telomeric sequences that form more stable G4 DNA than human telomeric sequences, induce more mutations. Stochastic telomere loss is seen in the premature aging disorder Werner Syndrome, which is caused by loss of the RecQ helicase protein WRN. We hypothesized that WRN deficiency leads to replication fork stalling and collapse due to G4 DNA formed by telomeric repeats resulting in deletions of DNA sequence. Shuttle vectors with a telomeric or control sequence were replicated in U2OS cells deficient or proficient for WRN. Replication of shuttle vectors in normal cells did not influence shuttle vector mutant frequencies, while WRN depleted cells exhibited elevated mutant frequencies for both telomeric and control vectors but the increase was significantly higher for the telomeric vector. We demonstrated that WRN is involved in suppressing mutagenesis in shuttle vectors with telomeric sequences. We are also testing DNA synthesis in plasmids through regions of single stranded DNA containing telomere repeats in WRN proficient and deficient cells. Public health significance: Shortened telomeres are associated with age related diseases such as heart disease, cancer and premature aging disorders. These assays will help us investigate factors that cause accelerated telomere loss with the goal of preventing or delaying disease.
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Details
Item Type: |
University of Pittsburgh ETD
|
Status: |
Unpublished |
Creators/Authors: |
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ETD Committee: |
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Date: |
29 June 2011 |
Date Type: |
Completion |
Defense Date: |
18 April 2011 |
Approval Date: |
29 June 2011 |
Submission Date: |
5 April 2011 |
Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
Institution: |
University of Pittsburgh |
Schools and Programs: |
School of Public Health > Human Genetics |
Degree: |
PhD - Doctor of Philosophy |
Thesis Type: |
Doctoral Dissertation |
Refereed: |
Yes |
Uncontrolled Keywords: |
DNA repair; DNA replication; G-quadruplex; Genomic instability; Mutagenesis; Telomere; Werner Syndrome |
Other ID: |
http://etd.library.pitt.edu/ETD/available/etd-04052011-214408/, etd-04052011-214408 |
Date Deposited: |
10 Nov 2011 19:34 |
Last Modified: |
15 Nov 2016 13:38 |
URI: |
http://d-scholarship.pitt.edu/id/eprint/6803 |
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