Mechanism for priming DNA synthesis by yeast DNA Polymerase α

Rajika L Perera,Joseph D Maman,Rubben Torella,Mairi L Kilkenny,Luca Pellegrini,Sebastian Klinge

doi:10.7554/elife.00482

Abstract

The DNA Polymerase α (Pol α)/primase complex initiates DNA synthesis in eukaryotic replication. In the complex, Pol α and primase cooperate in the production of RNA-DNA oligonucleotides that prime synthesis of new DNA. Here we report crystal structures of the catalytic core of yeast Pol α in unliganded form, bound to an RNA primer/DNA template and extending an RNA primer with deoxynucleotides. We combine the structural analysis with biochemical and computational data to demonstrate that Pol α specifically recognizes the A-form RNA/DNA helix and that the ensuing synthesis of B-form DNA terminates primer synthesis. The spontaneous release of the completed RNA-DNA primer by the Pol α/primase complex simplifies current models of primer transfer to leading- and lagging strand polymerases. The proposed mechanism of nucleotide polymerization by Pol α might contribute to genomic stability by limiting the amount of inaccurate DNA to be corrected at the start of each Okazaki fragment. DOI:http://dx.doi.org/10.7554/eLife.00482.001.

Highlights

Cellular organisms initiate DNA synthesis during genome duplication by the universal mechanism of RNA priming, the assembly of short RNA molecules on the unwound strands of the DNA helix by a specialized DNA-dependent RNA polymerase known as primase (Frick and Richardson, 2001; Kuchta and Stengel, 2010; DePamphilis and Bell, 2011)
We demonstrate that Polymerase α (Pol α) recognizes the intrinsic and induced conformation of the A-form RNA primer/ DNA template helix and that the resulting synthesis of B-form DNA forms the basis for a feedback mechanism of primer termination
The catalytic region of Pol α adopts the universal ‘right-hand’ DNA polymerase fold consisting of a palm domain harboring the active site, a fingers domain that interacts with the incoming nucleotide and a thumb domain that grips the primer-template duplex

Summary

Introduction

Cellular organisms initiate DNA synthesis during genome duplication by the universal mechanism of RNA priming, the assembly of short RNA molecules on the unwound strands of the DNA helix by a specialized DNA-dependent RNA polymerase known as primase (Frick and Richardson, 2001; Kuchta and Stengel, 2010; DePamphilis and Bell, 2011). The RNA primers are extended in an obligate 5′ to 3′ direction by the replicative DNA polymerases that synthesize the bulk of chromosomal DNA. The molecular apparatus responsible for initiation of DNA synthesis in eukaryotic replication is more complex. Reflecting its critical importance to DNA replication, the Pol α/primase complex is an integral component of the eukaryotic replisome (Calzada et al, 2005)

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Conclusion