Termination within oligo(dT) tracts in template DNA by DNA polymerase γ occurs with formation of a DNA triplex structure and is relieved by mitochondrial single-stranded DNA-binding protein

Abstract

Xenopus laevis DNA polymerase γ (pol γ) exhibits low activity on a poly(dT)-oligo(dA) primer-template. We prepared a single-stranded phagemid template containing a dT41 sequence to test the ability of pol γ to extend a primer through a defined oligo(dT) tract, pol γ terminates in the center of this dT41 sequence. This replication arrest is abrogated by addition of single-stranded DNA-binding protein or by substitution of 7-deaza-dATP for dATP. These features are consistent with the formation of a T·A*T DNA triplex involving the primer stem. Replication arrest occurs under conditions that permit highly processive DNA synthesis by pol γ. A similar replication arrest occurs for T7 DNA polymerase, which is also a highly processive DNA polymerase. These results suggest the possibility that DNA triplex formation can occur prior to dissociation of DNA polymerase. Primers with 3'-oligo(dA) termini annealed to a template with a longer oligo(dT) tract are not efficiently extended by pol γ unless single-stranded DNA-binding protein is added. Thus, one of the functions of single-stranded DNA-binding protein in mtDNA maintenance may be to enable pol γ to successfully replicate through dT-rich sequences.