UNWINDING OF THE 3RD 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 helic es, Intermolecular d(TC)(20). d(GA)(20). d(TC)(20) triplexes were gene rated by annealing, at pH 5.5, a linearized double-stranded plasmid co ntaining a d(TC)(27). d(GA)(27) tract with a P-32-labeled oligonucleot ide 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. Incubat ion with the T-ag helicase caused unwinding of the d(TC)(20) tract and consequent release of the oligonucleotide, while the plasmid molecule s remained double-stranded. ATP was required for this reaction and cou ld not be replaced by the non-hydrolyzable ATP analog AMP-PNP, T-ag di d 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 helic ase 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 thi rd strand, Unwinding of DNA triplexes by helicases may be required for processes such as DNA replication, transcription, recombination and r epair.