A conserved motif in the C-terminal tail of DNA polymerase α tethers primase to the eukaryotic replisome

Abstract

The DNA polymerase α-primase complex forms an essential part of the eukaryotic replisome. The catalytic subunits of primase and pol α synthesize composite RNA-DNA primers that initiate the leading and lagging DNA strands at replication forks. The physical basis and physiological significance of tethering primase to the eukaryotic replisome via pol α remain poorly characterized. We have identified a short conserved motif at the extreme C terminus of pol α that is critical for interaction of the yeast ortholog pol1 with primase. We show that truncation of the C-terminal residues 1452-1468 of Pol1 abrogates the interaction with the primase, as does mutation to alanine of the invariant amino acid Phe(1463). Conversely, a pol1 peptide spanning the last 16 residues binds primase with high affinity, and the equivalent peptide from human Pol α binds primase in an analogous fashion. These in vitro data are mirrored by experiments in yeast cells, as primase does not interact in cell extracts with pol1 that either terminates at residue 1452 or has the F1463A mutation. The ability to disrupt the association between primase and pol α allowed us to assess the physiological significance of primase being tethered to the eukaryotic replisome in this way. We find that the F1463A mutation in Pol1 renders yeast cells dependent on the S phase checkpoint, whereas truncation of Pol1 at amino acid 1452 blocks yeast cell proliferation. These findings indicate that tethering of primase to the replisome by pol α is critical for the normal action of DNA replication forks in eukaryotic cells.

FIGURE 1.

Evolutionary conservation of a short, conserved motif at the extreme C terminus of pol α. A, two views of the crystal structure of the yeast pol α CTD-B subunit complex (17). The polypeptide chains are drawn as orange (B) and blue (CTD) ribbons. The position of the natively unstructured tail at the C terminus of the CTD is indicated with blue dots in the top view panel. B, multiple sequence alignment of the C terminus of pol α (S.c.: Saccharomyces cerevisiae; S.p.: Schizosaccharomyces pombe; D.m.: Drosophila melanogaster; D.r.: Danio rerio; R.n.: Rattus norvegicus; H.s.: Homo sapiens). The position of the α-helix that represents the last structured segment of the CTD is indicated above the alignment. Asterisks mark conserved hydrophobic amino acids in a natively unstructured motif at the extreme C terminus of pol α.

FIGURE 2.

Gel filtration analysis of the heterotetrameric assembly reconstituted from heterodimeric Pri1-Pri2 primase and the Pol1 CTD-Pol12 complex. For each experiment, the chromatogram and the SDS-PAGE analysis (gels stained with Coomassie Blue) of the relevant fractions are shown.

FIGURE 3.

The C-terminal motif of pol α supports primase binding. A, peptides corresponding to wild-type and mutated versions of Pol1 residues 1453–1468 were expressed as GST fusions and tested in binding assays with yeast primase (SDS-PAGE stained with Coomassie Blue). B, same as A, but measuring the interaction of wild-type and mutated versions of amino acids 1445–1462 of human pol α with human primase. C, fluorescence anisotropy binding curves of human primase to fluorescein-labeled wild-type and F1455A mutant peptides spanning amino acids 1445–1462 of human pol α. D, cross-linking analysis of the interaction between human primase and a fluorescein-labeled peptide spanning amino acids 1445–1462 of human pol α. The result of the experiment is shown by SDS-PAGE (left panel, stained with Coomassie Blue) and UV fluorescence of the fluorescein-labeled peptide (right panel). A truncated version of human primase lacking the iron-sulfur domain (Prim2ΔCTD) was used to allow the unambiguous identification of the cross-linked primase subunit. The arrow indicates the position of the cross-linked peptide.

FIGURE 4.

The C-terminal motif of yeast pol α is essential for recruitment of primase to the replisome in vivo. A, extracts were made from asynchronous cultures of the indicated diploid yeast strains, before immunoprecipitation on anti-Myc beads. Proteins were separated by SDS-PAGE and digested with trypsin before detection of peptides by mass spectrometry (displayed as “spectral counts”). B, i, extracts from asynchronous cultures of the indicated diploid strains were subjected to immunoprecipitation on anti-HA beads. The indicated proteins were detected by immunoblotting using the corresponding antibodies as described under “Experimental Procedures.” ii, an equivalent experiment was performed using extracts of cells that had been treated with the cross-linking agent formaldehyde.

FIGURE 5.

Functional significance of tethering primase to the replisome in yeast cells. A, tetrad analysis of the meiotic progeny of a diploid strain with one copy of wild-type POL1 and one copy of pol1 1–1452. Scale bars, 20 μm. B, tetrad analysis of the meiotic progeny of a diploid strain with one copy of wild-type POL1 and one copy of pol1-F1463A. C, control and pol1-F1463A synchronized in G₁ phase with mating pheromone and then allowed to progress through a complete round of the cell cycle. Mating pheromone was added again at the 60-min time point so that cells completing cell division would not enter a second cell cycle. DNA content was monitored throughout the experiment by flow cytometry. D, tetrad analysis of the meiotic progeny of a diploid strain with the indicated genotype. Scale bars, 20 μm. E, a model for primase recruitment to the eukaryotic replisome. A conserved motif in the C-terminal tail of the catalytic subunit of pol α tethers primase to the replication fork. The names are those of the S. cerevisiae proteins.