THE SV40 LARGE T-ANTIGEN HELICASE CAN UNWIND 4-STRANDED DNA STRUCTURES LINKED BY G-QUARTETS

We describe a novel activity of the SV40 large T-ag helicase, the unwi nding of four stranded DNA structures linked by stacked G-quartets, na mely stacked groups of four guanine bases bound by Hoogsteen hydrogen bonds. The structures unwound by the helicase were of two types: (i) q uadruplexes comprising four parallel strands that were generated by an nealing oligonucleotides including clustered G residues in a buffer co ntaining Na+ ions, Each parallel quadruplex consisted of four oligonuc leotide molecules. (ii) Complexes comprising two parallel and two anti parallel strands that were generated by annealing the above oligonucle otides in a buffer containing K+ ions, Each antiparallel complex consi sted of two folded oligonucleotide molecules, Unwinding of these unusu al DNA structures by the T-ag was monitored by gel electrophoresis, Th e unwinding process required ATP and at least one single stranded 3'-t ail extending beyond the four stranded region, These data indicated th at 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 stra nded DNA into single strands, This helicase activity may affect proces ses such as recombination and telomere extension, in which four strand ed DNA could play a role.