Flexible tethering of primase and DNA Pol α in the eukaryotic primosome

Abstract

The Pol α/primase complex or primosome is the primase/polymerase complex that initiates nucleic acid synthesis during eukaryotic replication. Within the primosome, the primase synthesizes short RNA primers that undergo limited extension by Pol α. The resulting RNA–DNA primers are utilized by Pol δ and Pol ε for processive elongation on the lagging and leading strands, respectively. Despite its importance, the mechanism of RNA–DNA primer synthesis remains poorly understood. Here, we describe a structural model of the yeast primosome based on electron microscopy and functional studies. The 3D architecture of the primosome reveals an asymmetric, dumbbell-shaped particle. The catalytic centers of primase and Pol α reside in separate lobes of high relative mobility. The flexible tethering of the primosome lobes increases the efficiency of primer transfer between primase and Pol α. The physical organization of the primosome suggests that a concerted mechanism of primer hand-off between primase and Pol α would involve coordinated movements of the primosome lobes. The first three-dimensional map of the eukaryotic primosome at 25 Å resolution provides an essential structural template for understanding initiation of eukaryotic replication.

Structures of human primase reveal design of nucleotide elongation site and mode of Pol α tethering

“…Electron microscopy studies of a recombinant, unliganded version of the yeast Pol α/primase complex have shown that polymerase and primase reside in separate lobes of a flexible dumbbell-shaped particle (24). In agreement with the EM analysis, biochemical evidence shows that the interaction between primase and Pol α is mediated by the conserved C-terminal region of the catalytic subunit of Pol α (23,24).…”

“…Electron microscopy studies of a recombinant, unliganded version of the yeast Pol α/primase complex have shown that polymerase and primase reside in separate lobes of a flexible dumbbell-shaped particle (24). In agreement with the EM analysis, biochemical evidence shows that the interaction between primase and Pol α is mediated by the conserved C-terminal region of the catalytic subunit of Pol α (23,24). Furthermore, a conserved motif at the C terminus of the catalytic subunit of Pol α is necessary and sufficient for association with Significance DNA synthesis during duplication of the genome depends on primase, the DNA-dependent RNA polymerase that initiates nucleotide polymerization by assembling short RNA primers on the unwound template DNA.…”

Architecture of the Saccharomyces cerevisiae Replisome

“…Pol α-primase is a bilobed structure in which Pol1 binds to the B subunit and Pri1-Pri2 dimer through its CTD, and the CTD of Pol1 is connected to the polymerase region by a flexible stalk (Nunez-Ramirez et al 2011). Pol α-primase requires CMG for priming activity on both the leading and lagging strands during unwinding of the forked DNA in the presence of RPA (Georgescu et al 2015b).…”

The Eukaryotic Replisome Goes Under the Microscope

“…Crystal structures exist for individual subunits of Pol alpha [56–58], while single particle EM 3D reconstruction studies have provided the 3D architecture of the Pol alpha holoenzyme [59] (Figure 2A). The largest subunit, Pol1, contains the DNA polymerase as well as a CTD that is connected to the polymerase region by a flexible stalk [59]. The second largest subunit, Pol12, and the two smallest subunits that function together for primase activity, bind to the CTD at the end of the flexible stalk of Pol1.…”

“…The Pol1 polymerase subunit contains a CTD linked by a flexible tether to which the other subunits attach. Adapted from [59] by permission of Oxford University Press. (B) Surface representation of the C-half of S. cerevisiae Ctf4 trimer (magenta), with bound peptides modeled into the peptide binding pockets (blue).…”