Flexibility and distributive synthesis regulate RNA priming and handoff in human DNA polymerase α-primase

Abstract

DNA replication in eukaryotes relies on the synthesis of a ~30-nucleotide chimeric RNA/DNA primer strand through the dual action of the heterotetrameric polymerase α-primase (pol-prim) enzyme. Synthesis of an initial 7-10-nucleotides of RNA by primase is followed by intramolecular handoff of this primed template to pol α for extension of the primer by ~20 nucleotides of DNA. I investigated the structural mechanism for RNA primer synthesis using an integrative approach, providing evidence that RNA primer synthesis is governed by a combination of the high affinity and flexible linkage of the C-terminal domain of the primase regulatory subunit (PRIM2C) and the surprisingly low affinity of the primase catalytic domain (PRIM1) for substrate. Using a combination of small angle X-ray scattering and electron microscopy, I observed significant variability in the organization of PRIM2C and PRIM1 in the absence and presence of substrate, and that the population of structures with both PRIM2C and PRIM1 in a configuration aligned for synthesis is low. Crosslinking was used to visualize the orientation of PRIM2C and PRIM1 when engaged by substrate as observed by electron microscopy. Substrate binding affinity measurements for a series of pol-prim constructs showed that the PRIM1 catalytic domain does not bind the template or emergent RNA-primed templates with appreciable affinity. Together, these findings support a model of RNA primer synthesis in which generation of the nascent RNA strand and handoff of the RNA-primed template from primase to polymerase α is mediated by the high degree of inter-domain flexibility of pol-prim, the ready dissociation of PRIM1 from its substrate, and the much higher affinity of the POLA1 catalytic domain of polymerase α for full-length RNA-primed templates.