Dataset Information

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

ABSTRACT: Initiation of DNA synthesis in genomic duplication depends on primase, the DNA-dependent RNA polymerase that synthesizes de novo the oligonucleotides that prime DNA replication. Due to the discontinuous nature of DNA replication, primase activity on the lagging strand is required throughout the replication process. In eukaryotic cells, the presence of primase at the replication fork is secured by its physical association with DNA polymerase ? (Pol ?), which extends the RNA primer with deoxynucleotides. Our knowledge of the mechanism that primes DNA synthesis is very limited, as structural information for the eukaryotic enzyme has proved difficult to obtain. Here, we describe the crystal structure of human primase in heterodimeric form consisting of full-length catalytic subunit and a C-terminally truncated large subunit. We exploit the crystallographic model to define the architecture of its nucleotide elongation site and to show that the small subunit integrates primer initiation and elongation within the same set of functional residues. Furthermore, we define in atomic detail the mode of association of primase to Pol ?, the critical interaction that keeps primase tethered to the eukaryotic replisome.

SUBMITTER: Kilkenny ML

PROVIDER: S-EPMC3791718 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Kilkenny Mairi Louise ML Longo Michael Anthony MA Perera Rajika L RL Pellegrini Luca L

Proceedings of the National Academy of Sciences of the United States of America 20130916 40

Initiation of DNA synthesis in genomic duplication depends on primase, the DNA-dependent RNA polymerase that synthesizes de novo the oligonucleotides that prime DNA replication. Due to the discontinuous nature of DNA replication, primase activity on the lagging strand is required throughout the replication process. In eukaryotic cells, the presence of primase at the replication fork is secured by its physical association with DNA polymerase α (Pol α), which extends the RNA primer with deoxynucle ...[more]

PMID: 24043831

Similar Datasets

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.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets