Abstract
DNA polymerase alpha-primase (pol-prim) plays a central role in DNA replication in higher eukaryotes, initiating synthesis on both leading and lagging strand single-stranded DNA templates. Pol-prim consists of a primase heterodimer that synthesizes RNA primers, a DNA polymerase that extends them, and a fourth subunit, p68 (also termed B-subunit), that is thought to regulate the complex. Although significant knowledge about single-subunit primases of prokaryotes has accumulated, the functions and regulation of pol-prim remain poorly understood. In the SV40 replication model, the p68 subunit is required for primosome activity and binds directly to the hexameric viral helicase T antigen, suggesting a functional link between T antigen-p68 interaction and primosome activity. To explore this link, we first mapped the interacting regions of the two proteins and discovered a previously unrecognized N-terminal globular domain of p68 (p68N) that physically interacts with the T antigen helicase domain. NMR spectroscopy was used to determine the solution structure of p68N and map its interface with the T antigen helicase domain. Structure-guided mutagenesis of p68 residues in the interface diminished T antigen-p68 interaction, confirming the interaction site. SV40 primosome activity of corresponding pol-prim mutants decreased in proportion to the reduction in p68N-T antigen affinity, confirming that p68-T antigen interaction is vital for primosome function. A model is presented for how this interaction regulates SV40 primosome activity, and the implications of our findings are discussed in regard to the molecular mechanisms of eukaryotic DNA replication initiation.
Highlights
17112 JOURNAL OF BIOLOGICAL CHEMISTRY sive DNA synthesis
The sequence and structure of the C-terminal domain (CTD) of p68 are conserved in the B-subunits of DNA polymerases ␦ and ⑀; the p68CTD is tightly associated with the CTD of the p180 subunit [11,12,13]
Our data suggest a refined model for SV40 replication initiation, with potential roles for p68 N terminus (p68N) in facilitating pol-prim recruitment, correctly positioning pol-prim to bind and prime the template, and enhancing RNA primer extension into an RNA-DNA primer
Summary
Yeast Two-hybrid Assay—Coding sequences of p68 fragments were amplified by PCR and ligated into the NdeI/BamHI sites of pGBKT7 vector containing a Leu selection marker. Coding sequences of Tag fragments were amplified by PCR and ligated into the EcoRI/BamHI sites of pGADT7 vector containing a Trp selection marker (Clontech). Coding sequences of Tag fragments 303– 627 and 357– 627 were PCRamplified, cloned into the BamHI/EcoRI sites of the pGEX-2T expression vector (GE Healthcare), and verified by DNA sequencing. DNA Polymerase Assay—The polymerase activities of the WT and mutant pol-prim were assayed on a randomly primed poly(dA)-oligo(dT) template (20:1) as described previously [30]. Initiation of SV40 DNA Replication—Monopolymerase assays were carried out as described previously [24] except that pol-prim complexes and amounts varied as indicated in the figures and legends. Isothermal Titration Calorimetry—p68N constructs and Tag 303– 627 were buffer exchanged into 25 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, and either 1 mM dithiothreitol (for NMR) or 5 mM -mercaptoethanol. For the differential line broadening NMR experiment, 15N-1H HSQC spectra of 100 M p68N were recorded before and after the addition of 10 M unlabeled Tag 303– 627
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