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


RESULTS
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.


CONCLUSIONS
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.
Sauguet
Ludovic
L
Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.
Klinge
Sebastian
S
Perera
Rajika L
RL
Maman
Joseph D
JD
Pellegrini
Luca
L
D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D019915:DNA Primase / Q000737:chemistry*; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000737:chemistry*; D007506:Iron-Sulfur Proteins / Q000737:chemistry; D017510:Protein Folding; D021122:Protein Subunits; D012313:RNA / Q000096:biosynthesis; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*
D007506:Iron-Sulfur Proteins; D021122:Protein Subunits; C034477:RNA primers; D029701:Saccharomyces cerevisiae Proteins; D012313:RNA; D019915:DNA Primase; D004255:Deoxyribodipyrimidine Photo-Lyase
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