Template-dependent polymerization across discontinuous templates by the heterodimeric primase from the hyperthermophilic archaeon Sulfolobus solfataricus

Abstract

The eukaryotic-like primase from the hyperthermophilic archaeon Sulfolobus solfataricus (SsoPriSL) exhibits a range of activities including template-dependent de novo primer synthesis, primer extension and template-independent terminal nucleotidyl transfer using either rNTPs or dNTPs. Remarkably, the enzyme is able to synthesize products far longer than templates in vitro. Here we show that the long products resulted from template-dependent polymerization across discontinuous templates (PADT) by SsoPriSL. PADT was initiated through either primer synthesis or terminal transfer, and occurred efficiently on templates containing contiguous dCs. Template switching took place when the 3′-end of a growing strand synthesized on one template annealed to another template directly or following the terminal addition of nucleotides, and was subsequently extended on the new template. The key to PADT was the ability of SsoPriSL to promote strand annealing. SsoPriSL catalyzed PADT with either dNTPs or rNTPs as the substrates but preferred the latter. The enzyme remained active in PADT but became inefficient in primer synthesis in vitro when temperature was raised from 55°C to 70°C. Our results suggest that SsoPriSL is capable of bridging noncomplementary DNA ends and, therefore, may serve a role in double-strand DNA break repair in Archaea.