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Nucleic Acids Research, 1995, Vol. 23, No. 8 1292-1299
© 1995

MOLECULAR BIOLOGY

Formation of DNA triple helices inhibits DNA unwinding by the SV40 large T-antigen helicase

Mor Peleg, Vered Kopel, James A. Borowiec1 and Haim Manor*

Department of Biology, Technion-Israel of Technology Haifa 32-000, Israel 1Department of Biochemistry, New York University Medical Center New York, NY 10016, USA

* To whom correspondence should be addressed

Received January 24, 1995. Revised March 6, 1995. Accepted March 6, 1995.

Previous studies have indicated that d(TC)n.d(GA)n microsateilites may serve as arrest signals for mammalian DNA replication through the ability of such sequences to form DNA triple helices and thereby inhibit replication enzymes. To further test this hypothesis, we examined the ability of d(TC)i.d(GA)i.d(TC)i triplexes to inhibit DNA unwinding in vitro by a model eukaryotic DNA helicase, the SV40 large T-antigen. DNA substrates that were able to form triplexes, and non-triplex-forming control substrates, were tested. We found that the presence of DNA triplexes, as assayed by endonuclease S1 and osmium tetroxide footprinting, significantly inhibitedDNA unwinding by T-antigen. Strong inhibition was observed not only at acidic pH values, in which the triplexes were most stable, but also at physiological pH values in the range 6.9–7.2. Little or no inhibition was detected at pH 8.7. Based on these results, and on previous studies of DNA polymerases, we suggest that DNA triplexes may form in vivo and cause replication arrest through a dual inhibition of duplex unwinding by DNA helicases and of nascent strandsynthesis by DNA polymerases. DNA triplexes also have the potential to inhibit recombination and repair processes in whichhelicases and polymerases are involved.


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