Crystal Structure of the Human Primase

Andrey G Baranovskiy,Yinbo Zhang,Yoshiaki Suwa,Nigar D Babayeva,Jianyou Gu,Youri I Pavlov,Tahir H Tahirov

doi:10.1074/jbc.m114.624742

Andrey G Baranovskiy, Yinbo Zhang + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m114.624742

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Abstract

DNA replication in bacteria and eukaryotes requires the activity of DNA primase, a DNA-dependent RNA polymerase that lays short RNA primers for DNA polymerases. Eukaryotic and archaeal primases are heterodimers consisting of small catalytic and large accessory subunits, both of which are necessary for RNA primer synthesis. Understanding of RNA synthesis priming in eukaryotes is currently limited due to the lack of crystal structures of the full-length primase and its complexes with substrates in initiation and elongation states. Here we report the crystal structure of the full-length human primase, revealing the precise overall organization of the enzyme, the relative positions of its functional domains, and the mode of its interaction with modeled DNA and RNA. The structure indicates that the dramatic conformational changes in primase are necessary to accomplish the initiation and then elongation of RNA synthesis. The presence of a long linker between the N- and C-terminal domains of p58 provides the structural basis for the bulk of enzyme's conformational flexibility. Deletion of most of this linker affected the initiation and elongation steps of the primer synthesis.

Highlights

DNA primase synthesizes RNA primers and is indispensable for genome replication
Such flexibility was observed in the structures of human and Sulfolobus solfataricus (Sso) primases with a deleted C-terminal domain of the large subunit and can play a role in PrimPol ␣ function [16, 26]
The proposed nucleoside triphosphate (NTP)/DNA-binding interface of p58C [17, 18] is facing the p49 active site (Fig. 1B), which may facilitate their cooperation during RNA synthesis in the cis-position

Summary

Background

DNA primase synthesizes RNA primers and is indispensable for genome replication. Results: We present a crystal structure of the intact human primase at 2.65 Å resolution. The first information about an overall three-dimensional architecture of eukaryotic primases was derived from the crystal structures of distantly related archaeal primases [23], including the structures of the Pyrococcus furiosus (Pfu) and Pyrococcus horikoshi (Pho) primase catalytic subunits, and the structure of the Sulfolobus solfataricus (Sso) primase catalytic subunit in complex with the N-terminal domain of its large subunit (24 –26) These data and mapping of the active site of mouse p49 by site-directed mutagenesis allowed determination of the positions of three invariant catalytic aspartates [27]. The crystal structures of the catalytic subunit of yeast primase and the human p49-p58(1–253) complex have been determined [16, 28] These results confirmed the early idea proposing similar organization of the active site in eukaryotic and archaeal primases and the role of three catalytic aspartates in the coordination of two divalent ions and NTP [23, 24]. In a set of structure-based functional assays, we clarified the mechanism of the primer length counting and confirmed that proper length of the linker connecting the N- and C-terminal domains of p58 is required for RNA primer synthesis

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION