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Nucleic Acids Research, 1991, Vol. 19, No. 7 1633-1638
© 1991

MOLECULAR BIOLOGY

Photofootprinting of DNA triplexes

Victor I. Lyamichev1, Oleg N. Voloshin, Maxim D. Frank-Kamenetskii1 and Valery N. Soyfer*

Department of Biology, George Mason University Fairfax, VA 22030, USA 1Institute of Molecular Genetics of the USSR Academy of Sciences Moscow 123182, USSR

* To whom correspondence should be addressed

Received November 12, 1990. Revised February 28, 1991. Accepted February 28, 1991.

We have used a photofootprinting assay to study intermolecular and intramolecular DNA triplexes. The assay is based on the fact that the DNA duplex is protected against photodamage (specifically, against the formation of the (6–4) pyrimidine photoproducts) within a triplex structure. We have shown that this is the case for PyPuPu (YRR) as well as PyPuPy (YRY) triplexes. Using the photofootprinting assay, we have studied the triplex formation under a variety of experimentally defined conditions. At acid pH, d(C)ncd(G)ncd(C)n and d(CT)ncd(GA)ncd(CT)n triplexes are detected by this method. The d(CT)ncd(GA)ncd(CT)n triplexes are additionally stabilized by divalent cations and spermidine. PyPuPu triplexes are pH-independent and are stabilized by divalent cations, such as Mg++ and Zn++. The effect depends on the type of cation and on the DNA sequence. The d(CT)ncd(GA)ncd(GA)n triplex is stabilized by Zn++ but not by Mg++ whereas the d(C)ncd(G)ncd(G)n triplex is stabilized by Mg++. In H-DNA, virtually the entire pyrimidine chain is protected against photodimerization, whereas only half of the pyrimidine chain participating in a triplex is protected in the CGG intramolecular triplex.


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