Unwinding of the third strand of a DNA triple helix, a novel activity of the SV40 large T-antigen helicase

We present experiments indicating that the SV40 large T-antigen (T-ag) helicase is capable of unwinding the third strand of DNA triple helices. Intermolecular d(TC)(20)d(GA)(20)d(TC)(20) triplexes were generated by annealing, at pH 5.5, a linearized double-stranded plasmid containing a d(TC)(27).d(GA)27 tract with a (32)P-labeled oligonucleotide consisting of a d(TC)(20) tract flanked by a sequence of 15 nt at the 3'-end. The triplexes remained stable at pH 7.2, as determined by agarose gel electrophoresis and dimethyl sulfate footprinting. Incubation with the T-ag helicase caused unwinding of the d(TC)(20) tract and consequent release of the oligonucleotide, while the plasmid molecules remained double-stranded. ATP was required for this reaction and could not be replaced by the non-hydrolyzable ATP analog AMP-PNP. T-ag did not unwind similar triplexes formed with oligonucleotides containing a d(TC)(20) tract and a 5' flanking sequence or no flanking sequence. These data indicate that unwinding of DNA triplexes by the T-ag helicase must be preceded by binding of the helicase to a single-stranded 3' flanking sequence, then the enzyme migrates in a 3'--> 5' direction, using energy provided by ATP hydrolysis, and causes release of the third strand. Unwinding of DNA triplexes by helicases may be required for processes such as DNA replication, transcription, recombination and repair.