Initiation of DNA replication by a third parallel DNA strand bound in a triple-helix manner leads to strand invasion.

According to current knowledge, DNA polymerases accommodate only two polynucleotide strands in their catalytic site: the template and the primer to be elongated. Here we show that in addition to these two polynucleotide strands, HIV-1 and AMV reverse transcriptases, human DNA polymerases beta, gamma, and lambda, and the archaebacterial Dpo4 can elongate 10-nucleotide primers bound in a triple-helix manner to hairpin duplex DNA tethered by a few thymidine residues. The elongation occurs when the primer is parallel to the homologous strand.