Delving into polymerase α-primase dynamics

Posted by daviskd2 on Friday, January 5, 2024 in News.

In eukaryotes, DNA replication relies on the synthesis of a chimeric RNA/DNA primer by the heterotetrameric polymerase α-primase (pol-prim) enzyme. The primers generated by pol-prim consist of 7-10 ribonucleotides followed by ~20 deoxyribonucleotides and are generated by distinct active sites in the primase and polymerase subunits. Both primase and polymerase α consist of a catalytic and a regulatory subunit: PRIM1 and PRIM2 for primase and POLA1 and POLA2 for polymerase α. PRIM2 is further divided into PRIM2N, which acts as a scaffold between primase and pol α subunits, and PRIM2C, which regulates both DNA and RNA synthesis. While the necessity of PRIM2C for RNA primer synthesis has been well established biochemically, a direct structural characterization of the priming complex during RNA catalysis has not been conducted.

In this recent study, Johnny Cordoba, recent Chazin lab graduate, and Elwood Mullins, a research assistant professor in the Eichman lab, et al. used a combination of structural and biophysical techniques to investigate the role of PRIM2C in primer synthesis. Small-angle X-ray scattering (SAXS) and negative stain EM demonstrated that significant interdomain flexibility exists within the entire pol-prim heterotetramer and PRIM2C especially adopts a wide range of orientations relative to the tetramer core. Microscale thermophoresis also determined that PRIM1, the catalytic domain, did not bind substrate with any appreciable affinity.

Taken together, these data suggest a model in which PRIM2C acts as both a processivity factor and length regulator in RNA synthesis. Firstly, tight binding between PRIM2C and the substrate along with inter-domain flexibility of pol-prim allows for PRIM1 association and disassociation during distributive synthesis of the RNA primer. Secondly, the ready dissociation of PRIM1 from its substrate and higher affinity of the POLA1 catalytic domain for full-length RNA-primed template facilitate handoff of the template from primase to polymerase α at specific lengths of 7-10 ribonucleotides. This model sheds new light on the intricacies of RNA primer synthesis and provides framework for future studies into the interplay of components in the replisome.

Read more about pol-prim dynamics here! ~Cameron I.Cohen