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

Abstract

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.

Interference Footprinting Analysis of Telomerase Elongation Complexes

“…This result is compatible with a previous observation that formation of a Watson-Crick base pair between residue N5 in the primer and residue A51 in the RNA template is less essential for the extension reaction than formation of a base pair between residue N4 in the primer and A50 in the template (19). Figure 4B shows interference footprinting assays performed with this oligonucleotide, using formic acid and KMnO 4 . It can be seen that modifications of residues G2, G3, and A5 strongly interfered with the extension of the primer, whereas the interference due to modifications of residues G6 and G7 was more moderate.…”

“…Inspection of the KMnO 4 footprint of oligo tel3 reveals that the interference caused by modification of residue T4 was much less substantial than that observed in the KMnO 4 footprint of oligo tel1. A similar observation was made in a KMnO 4 footprinting assay of an additional variant of oligo tel1 in which residue T5 was deleted (not shown). Apparently, the absence of T5 affected the interactions of these primers with the enzyme so that the role of T4 in the reaction became less significant.…”

Inhibition of DNA Replication and Induction of S Phase Cell Cycle Arrest by G-rich Oligonucleotides

“…Therefore, we investigated the effects of GRO29A on the replication-associated proteins most likely to be modulated. These were the SV40 large T antigen, which is known to bind to and unwind G-quartet structures (42), and RPA, which is known to bind to nucleolin (20), the putative GRO-binding protein (9). Although we could observe the interaction between nucleolin and RPA, we found that this was not significantly affected by the presence of GROs.…”

“…The ability of T antigen to unwind structures containing G-quartets has been documented previously (42); therefore, we postulated that the presence of an excess of G-quartet containing oligonucleotide could sequester the helicase activity and prevent template unwinding and replication. To test this hypothesis we carried out T antigen helicase assays in the presence of various oligonucleotides.…”

“…Many of the proteins that are required for efficient replication, both in the in vitro system and in cells, have now been identified (39 -41). Based on the existing literature, we believe the most likely replication-associated proteins to be modulated GRO29A are replication protein A (RPA), which is known to bind to nucleolin (20), or the SV40 large T antigen, which can bind to G-quartet structures (42).…”