Publication Date

2008-01-03

Availability

Open access

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Biochemistry and Molecular Biology (Medicine)

Date of Defense

2007-11-15

First Committee Member

Walter A. Scott - Committee Chair

Second Committee Member

Mary Lou King - Committee Member

Third Committee Member

Mathias G. Lichtenheld - Committee Member

Fourth Committee Member

Terace M. Fletcher - Mentor

Fifth Committee Member

Juan Ausio - Outside Committee Member

Abstract

Human telomeric DNA consists of tandem repeats of the sequence 5'-d(TTAGGG)-3' assembled into a nucleoprotein complex that functions to protect the ends of chromosomes. Such guanine-rich DNA is capable of forming a variety of G-quadruplexes, which in turn, can have varying functional consequences on telomere maintenance. G-quadruplex stabilizing ligands have been shown to induce chromosome end-to-end fusions, senescence and apoptosis, effects similar to the expression of a dominant-negative TTAGGG Repeat Factor 2 (TRF2). With this in mind, we analyzed the effect of sequence and length of human telomeric DNA, as well as cation conditions on G-quadruplex formation by native polyacrylamide gel electrophoresis and circular dichroism. We show that K+ and Sr2+ can induce human telomeric DNA to form both inter- and intramolecular structures. Circular dichroism results suggest that the structures in K+ were a mix of parallel and antiparallel G-quadruplexes, while Sr2+ induced only parallel-stranded structures. We also found that TRF2, a protein essential for telomere maintenance, affects G-quadruplex structure. These structures serve as useful models to study the effects of G-quadruplexes on the activities of telomeric proteins, like TRF2, from human cells. The G-strand overhang at the ends of telomeres may periodically adopt at least some of these quadruplex conformations, which could subsequently affect protein binding and telomere function. TRF2, a protein essential for telomere maintenance, is not known to bind single-strand (ss) DNA, work performed in the lab suggested that the type of 3'-overhang in telomeric DNA ss/ds-junctions affects TRF2-binding. Specifically, preventing G-quadruplex formation by changing the overhang sequence from 5'-d(TTAGGG)4-3', to 5'-dTTAGGG(TTAGAG)2TTAGGG-3', reduced TRF2 recruitment to the ss/ds-junction from HeLa cell extracts. Using the same techniques as above, we show that the N-terminal basic domain of TRF2 in K+ induces a switch from the mixed parallel/antiparallel-stranded G-quadruplexes usually stabilized by K+-alone, to parallel-stranded G-quadruplexes. Interestingly, it also promotes intermolecular parallel G-quadruplex formation on non-quadruplex, single-stranded intermediates, but will not induce a switch from an antiparallel to a parallel G-quadruplex in Na+. These results are the first to demonstrate specific TRF2 G-quadruplex interactions, suggesting a novel mechanism for TRF2 recognition of the ds/ss junction of telomeres.

Keywords

TRF2; Telomere; G-quadruplex

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