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Mutagenic Potential of Telomeric Repeats and the Role of Werner Syndrome Helicase Protein in Facilitating Telomeric DNA Replication

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 (Unpublished)

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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:
CreatorsEmailPitt UsernameORCID
Damerla, Rama Raorama.damerla@gmail.com
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairGollin, Susannesgollin@hgen.pitt.eduGOLLIN
Committee CoChairOpresko, Patriciaplo4@pitt.eduPLO4
Committee MemberNiedernhofer, Lauraniedernh@pitt.eduNIEDERNH
Committee MemberBarmada, Michaelbarmada@pitt.eduBARMADA
Committee MemberSobol, Robertrws9@pitt.eduRWS9
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: Graduate 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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