Priming DNA Replication from Triple Helix Oligonucleotides: Possible Threestranded DNA in DNA Polymerases
Figure 2
Determination of the orientation of the THP on a double-stranded DNA. The polypurine and polypyrimidine strands (50 pmol) were annealed at equimolar amounts. 5 pmoles of 32P-end-labelled primers were tested with their corresponding binding site ending with a C/C mismatch in a replication assay with 5.10–8 M human DNA polymerase β. The Reaction media contained either 0.2 mM dGTP, and dATP or 0.2 mM dGTP, dCTP and dTTP. If the primer (A)10 was parallel to the homologous strand, then, in the presence of dCTP and dTTP, there is synthesis of a 50 nt-long product. If the orientation was antiparallel, then a 30 nt-long product would be synthesized in the presence of dATP and dGTP. Conversely, if the d(T)10 primer is parallel to its homologous primer binding site, then a product of 30 nt-long would be observed in the presence of dGTP, dCTP, and dTTP, while no product was observed with dATP and dGTP [16].
