Shared active site architecture between the large subunit of eukaryotic primase and DNA photolyase

Abstract

Background: DNA synthesis during replication relies on RNA primers synthesised by the primase, a specialised DNA-dependent RNA polymerase that can initiate nucleic acid synthesis de novo. In archaeal and eukaryotic organisms, the primase is a heterodimeric enzyme resulting from the constitutive association of a small (PriS) and large (PriL) subunit. The ability of the primase to initiate synthesis of an RNA primer depends on a conserved Fe-S domain at the C-terminus of PriL (PriL-CTD). However, the critical role of the PriL-CTD in the catalytic mechanism of initiation is not understood.

Methodology/principal findings: Here we report the crystal structure of the yeast PriL-CTD at 1.55 A resolution. The structure reveals that the PriL-CTD folds in two largely independent alpha-helical domains joined at their interface by a [4Fe-4S] cluster. The larger N-terminal domain represents the most conserved portion of the PriL-CTD, whereas the smaller C-terminal domain is largely absent in archaeal PriL. Unexpectedly, the N-terminal domain reveals a striking structural similarity with the active site region of the DNA photolyase/cryptochrome family of flavoproteins. The region of similarity includes PriL-CTD residues that are known to be essential for initiation of RNA primer synthesis by the primase.

Conclusion/significance: Our study reports the first crystallographic model of the conserved Fe-S domain of the archaeal/eukaryotic primase. The structural comparison with a cryptochrome protein bound to flavin adenine dinucleotide and single-stranded DNA provides important insight into the mechanism of RNA primer synthesis by the primase.

Figure 1. Crystal structure of yeast PriL-CTD at 1.55 Å.

(A) Ribbon diagram of the PriL-CTD. The larger N-terminal domain is coloured in red and the smaller C-terminal domain in blue. The Fe-S cluster cofactor is shown in space-fill representation; the Fe and S atoms are in red and yellow, respectively. (B) Topology diagram of the PriL-CTD; the numbering and residue span of each alpha helix is indicated. The cysteine residues that ligate the Fe-S cluster are explicitly indicated. Colouring as in panel (A). (C) Close-up view of the Fe-S cluster. Hydrophobic residues surrounding the Fe-S cluster are shown. The main-chain trace of the PriL-CTD is shown as a thin tube, whereas the Fe-S cluster and side chains are drawn as sticks.

Figure 2. Structural similarity of the PriL-CTD and DNA photolyase.

(A) Cross-eye stereo-diagram of the N-terminal domain of PriL-CTD superimposed on the active site region of cryptochrome Cry3 from A. thaliana, bound to FAD and ssDNA. The PriL-CTD is drawn as a thin tube in blue, the DNA photolyase in green. FAD and ssDNA are shown as sticks, coloured according to element type. (B) Structure-based alignment of a representative set of primase and photolyase sequences spanning the region of structural homology (GenBank protein ID: S. cerevisiae, AAA34900; D. melanogaster, AAG01548; A. thaliana, AAM61309; X. laevis; AAH88966; H. sapiens, AAH64931; C. elegans, CAB03469; D. melanogaster, BAA12067; A. thaliana, BAC65244; X. laevis, AAI69685; T. thermophilus, AAS82388; E. coli, CAR11998; S. tokodaii, BAB65903). Absolutely conserved positions are highlighted in red, strongly conserved positions in yellow and weakly conserved positions in pale yellow. The alpha helical elements in the alignment are indicated above the sequences. (C) Cartoon of the heterodimeric primase, showing a possible model for the essential role of the PriL-CTD in initiation of RNA primer synthesis. According to the model, the PriL-CTD would assist the catalytic subunit PriS in the simultaneous binding of the two initial ribonucleotides and in promoting their Watson-Crick base pairing at the initiation site on the template DNA.

Figure 3. Structural features of the yeast PriL-CTD.

(A) Amino acid conservation mapped on the crystal structure of the PriL-CTD. The evolutionary conservation analysis of surface residues was performed with the ConSurf server , based on 49 sequences of eukaryotic primases. Degree of conservation is shown by colour range, from magenta (highest conservation) to cyan (lowest). The structure is shown in space fill representation. Highly conserved residues that might be important for the functional role of the PriL-CTD are indicated. The ssDNA molecule of the DASH cryptochrome 3 co-crystal (PDB id: 2VTB), superimposed on the PriL-CTD, is shown in stick representation. (B) Electrostatic potential of the PriL-CTD, mapped on its solvent-accessible surface at contouring levels of ±5 kT. Positive charge is in blue, negative charge in red. The potential was calculated using APBS in PyMol (http://www.pymol.org/). The ssDNA is depicted as in panel B. (C) and (D) Structural details of PriL-CTD residues known to have important functional roles in primase function. Panel C depicts Arg 355 and His 401, panel D shows Lys 363. Please see text for the functional interpretation of their role. The side chains are shown as thick or thin sticks, the helices as cylinders. The ssDNA from the cryptochrome 3 co-crystal structure is also shown in yellow. Polar interactions involving Lys 363 are drawn as dashed yellow lines.

Figure 4. The PriL-CTD binds DNA.

(A) Fluorescence anisotropy curves of fluorescein-labelled DNA in the presence of increasing amounts of PriL-CTD. Each data point is the average of three independent measurements. (B) Fluorescence anisotropy curves of fluorescein-labelled DNA in the presence of full-length (WT) or truncated primase, missing the PriL-CTD (ΔCTD). Each data point is the average of three independent measurements. (C) Cartoon of the possible evolutionary history of the PriL. The PriL might have derived from a smaller Fe-S protein (PriLp: PriL precursor; ISC: Iron-Sulfur Cluster) with moderate affinity for DNA, reflecting an unknown function in nucleic acid metabolism. Recruitment of PriLp to a ‘prim’-fold polymerase, the ancestor of current PriS, would have allowed initiation of DNA synthesis during replication. Over time, the PriLp would have become incorporated in a constitutive heterodimer with PriS.