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Primase-polymerases: how to make a primer from scratch.

ABSTRACT: To pass on genetic information to the next generation, cells must faithfully replicate their genomes to provide copies for each daughter cell. To synthesise these duplicates, cells employ specialised enzymes called DNA polymerases, which rapidly and accurately replicate nucleic acid polymers. However, most polymerases lack the ability to directly initiate DNA synthesis and required specialised replicases called primases to make short polynucleotide primers, from which they then extend. Replicative primases (eukaryotes and archaea) belong to a functionally diverse enzyme superfamily known as Primase-Polymerases (Prim-Pols), with orthologues present throughout all domains of life. Characterised by a conserved catalytic Prim-Pol domain, these enzymes have evolved various roles in DNA metabolism, including DNA replication, repair, and damage tolerance. Many of these biological roles are fundamentally underpinned by the ability of Prim-Pols to generate primers de novo. This review examines our current understanding of the catalytic mechanisms utilised by Prim-Pols to initiate primer synthesis.

SUBMITTER: Bainbridge LJ

PROVIDER: S-EPMC10345425 | biostudies-literature | 2023 Jul

REPOSITORIES: biostudies-literature

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Primase-polymerases: how to make a primer from scratch.

Bainbridge Lewis J LJ Zabrady Katerina K Doherty Aidan J AJ

Bioscience reports 20230701 7

To pass on genetic information to the next generation, cells must faithfully replicate their genomes to provide copies for each daughter cell. To synthesise these duplicates, cells employ specialised enzymes called DNA polymerases, which rapidly and accurately replicate nucleic acid polymers. However, most polymerases lack the ability to directly initiate DNA synthesis and required specialised replicases called primases to make short polynucleotide primers, from which they then extend. Replicati ...[more]

PMID: 37358261

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Project description:The eukaryotic polymerase α (Pol α) is a dual-function DNA polymerase/primase complex that synthesizes an RNA-DNA hybrid primer of 20-30 nucleotides for DNA replication. Pol α is composed of Pol1, Pol12, Primase 1 (Pri1), and Pri2, with Pol1 and Pri1 containing the DNA polymerase activity and RNA primase activity, respectively, whereas Pol12 and Pri2 serve a structural role. It has been unclear how Pol α hands over an RNA primer made by Pri1 to Pol1 for DNA primer extension, and how the primer length is defined, perhaps due to the difficulty in studying the highly mobile structure. Here we report a comprehensive cryo-EM analysis of the intact 4-subunit yeast Pol α in the apo, primer initiation, primer elongation, RNA primer hand-off from Pri1 to Pol1, and DNA extension states in a 3.5 Å - 5.6 Å resolution range. We found that Pol α is a three-lobed flexible structure. Pri2 functions as a flexible hinge that holds together the catalytic Pol1-core, and the noncatalytic Pol1 CTD that binds to Pol 12 to form a stable platform upon which the other components are organized. In the apo state, Pol1-core is sequestered on the Pol12-Pol1-CTD platform, and Pri1 is mobile perhaps in search of a template. Upon binding a ssDNA template, a large conformation change is induced that enables Pri1 to perform RNA synthesis, and positions Pol1-core to accept the future RNA primed site 50 Å upstream of where Pri1 binds. We reveal in detail the critical point at which Pol1-core takes over the 3'-end of the RNA from Pri1. DNA primer extension appears limited by the spiral motion of Pol1-core while Pri2-CTD stably holds onto the 5' end of the RNA primer. Since both Pri1 and Pol1-core are attached via two linkers to the platform, primer growth will produce stress within this "two-point" attachment that may limit the length of the RNA-DNA hybrid primer. Hence, this study reveals the large and dynamic series of movements that Pol α undergoes to synthesize a primer for DNA replication.

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Primase-polymerases: how to make a primer from scratch.

Publications

Primase-polymerases: how to make a primer from scratch.

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