Title

The Outer Limits: Telomere maintenance by TRF2 and G-quadruplex DNA structures

Date of Award

2007

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

First Committee Member

Terace M. Fletcher, Committee Chair

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. Although the structures of short telomeric oligonucleotides have been carefully examined, few studies have focused on longer telomeric DNA, which mimics the very ends of telomeres. 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, which binds towards the end of the double-strand (ds) telomere region just upstream of the G-strand overhang, is considered one of the key proteins in telomere protection and regulation. While TRF2 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(TTAG AG)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-quadruplexcs. 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. They suggest a novel mechanism for TRF2 recognition of the double-strand/single-strand junction of telomeres, where the myb-like domain binds to the double-strand DNA and the N-terminal basic domain interacts with the overhang, stabilizing the interaction. This model has implications for TRF2 communication with the very terminus of the telomere and for stimulation of strand invasion proposed to stabilize t-loop structures.

Keywords

Biology, Molecular; Chemistry, Biochemistry

Link to Full Text

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