VC++ version of program for coauthor paper http://www.ncbi.nlm.nih.gov/pubmed/18158301
Nucleic Acids Res. 2008 Mar;36(4):1200-8. Epub 2007 Dec 23.
G-quadruplex preferentially forms at the very 3' end of vertebrate telomeric DNA.
Tang J, Kan ZY, Yao Y, Wang Q, Hao YH, Tan Z.
Abstract
Human chromosome ends are protected with kilobases repeats of TTAGGG. Telomere DNA shortens at replication. This shortening in most tumor cells is compensated by telomerase that adds telomere repeats to the 3' end of the G-rich telomere strand. Four TTAGGG repeats can fold into G-quadruplex that is a poor substrate for telomerase. This property has been suggested to regulate telomerase activity in vivo and telomerase inhibition via G-quadruplex stabilization is considered a therapeutic strategy against cancer. Theoretically G-quadruplex can form anywhere along the long G-rich strand. Where G-quadruplex forms determines whether the 3' telomere end is accessible to telomerase and may have implications in other functions telomere plays. We investigated G-quadruplex formation at different positions by DMS footprinting and exonuclease hydrolysis. We show that G-quadruplex preferentially forms at the very 3' end than at internal positions. This property provides a molecular basis for telomerase inhibition by G-quadruplex formation. Moreover, it may also regulate those processes that depend on the structure of the very 3' telomere end, for instance, the alternative lengthening of telomere mechanism, telomere T-loop formation, telomere end protection and the replication of bulky telomere DNA. Therefore, targeting telomere G-quadruplex may influence more telomere functions than simply inhibiting telomerase.
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Quantitative footprint of G-quadruplex
DNA treatment and gel electrophoresis was carried out essentially as previously described (25) using 32P-labeled oligonucleotide (∼20 000 cpm, 5 ng). The oligonucleotides were treated with DMS for 30, 60, 90 s, respectively, in the presence or absence of 150 mM K+. DNA cleavage products were resolved by gel electrophoresis, autoradiographed on a Typhoon phosphor imager (Amersham Biosciences, Sweden) and the intensity of the bands corresponding to attacks at each G triplet quantified with the software ImageQuant 5.2.
The equations describing the production of DNA fragments were solved by the standard Steepest Descent Method (27, 28). The algorithm was implemented with Visual C++ in a stand-alone application. In this program, the sum of the intensity of the bands produced by cleavage at each G triplet can be input through a friendly graphical interface and the parameters, R, Qi and p, can be obtained simultaneously with an optional initial value calculation.