The SV40 large T-antigen helicase can unwind four stranded DNA structures linked by G-quartets.

We describe a novel activity of the SV40 large T-ag helicase, the unwinding of four stranded DNA structures linked by stacked G-quartets, namely stacked groups of four guanine bases bound by Hoogsteen hydrogen bonds. The structures unwound by the helicase were of two types: (i) quadruplexes comprising four parallel strands that were generated by annealing oligonucleotides including clustered G residues in a buffer containing Na+ions. Each parallel quadruplex consisted of four oligonucleotide molecules. (ii) Complexes comprising two parallel and two antiparallel strands that were generated by annealing the above oligonucleotides in a buffer containing K+ions. Each antiparallel complex consisted of two folded oligonucleotide molecules. Unwinding of these unusual DNA structures by the T-ag was monitored by gel electrophoresis. The unwinding process required ATP and at least one single stranded 3'-tail extending beyond the four stranded region. These data indicated that the T-ag first binds the 3'-tail and moves in a 3'-->5'direction, using energy provided by ATP hydrolysis; then it unwinds the four stranded DNA into single strands. This helicase activity may affect processes such as recombination and telomere extension, in which four stranded DNA could play a role.