title: 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.
  
  
  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.
authors.affiliation: Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Ludovic
authors.initials: L
authors.lastname: Sauguet
authors.forename: Sebastian
authors.initials: S
authors.lastname: Klinge
authors.forename: Rajika L
authors.initials: RL
authors.lastname: Perera
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Maman
authors.forename: Luca
authors.initials: L
authors.lastname: Pellegrini
mesh_terms: 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*
chemical_list: D007506:Iron-Sulfur Proteins; D021122:Protein Subunits; C034477:RNA primers; D029701:Saccharomyces cerevisiae Proteins; D012313:RNA; D019915:DNA Primase; D004255:Deoxyribodipyrimidine Photo-Lyase
references.citation: J Biol Chem. 1993 Dec 15;268(35):26179-89
references.pmid: 8253737
references.citation: Mol Cell. 2008 May 9;30(3):259-60
references.pmid: 18471969
references.citation: Trends Biochem Sci. 2000 Nov;25(11):572-6
references.pmid: 11084371
references.citation: Eur J Biochem. 1986 Dec 1;161(2):435-40
references.pmid: 3536513
references.citation: Proc Natl Acad Sci U S A. 1991 May 1;88(9):3877-81
references.pmid: 2023935
references.citation: Curr Opin Struct Biol. 2006 Feb;16(1):51-9
references.pmid: 16427270
references.citation: Methods Mol Biol. 2008;426:419-35
references.pmid: 18542881
references.citation: Proc Natl Acad Sci U S A. 2008 Dec 30;105(52):21023-7
references.pmid: 19074258
references.citation: Biochemistry. 1999 Sep 28;38(39):12899-907
references.pmid: 10504261
references.citation: Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
references.pmid: 15572765
references.citation: Chem Rev. 2003 Jun;103(6):2203-37
references.pmid: 12797829
references.citation: Nat Struct Mol Biol. 2005 Dec;12(12):1137-44
references.pmid: 16273105
references.citation: Curr Opin Struct Biol. 2009 Jun;19(3):277-85
references.pmid: 19487120
references.citation: Nat Struct Mol Biol. 2007 Sep;14(9):875-7
references.pmid: 17704817
references.citation: J Mol Biol. 1993 Jan 5;229(1):105-24
references.pmid: 7678431
references.citation: J Mol Biol. 2004 Aug 27;341(5):1145-59
references.pmid: 15321712
references.citation: Acta Crystallogr D Biol Crystallogr. 1997 May 1;53(Pt 3):240-55
references.pmid: 15299926
references.citation: Mol Cell Biol. 1989 Jul;9(7):3081-7
references.pmid: 2528682
references.citation: Nucleic Acids Res. 2007 Jul;35(Web Server issue):W375-83
references.pmid: 17452350
references.citation: J Biol Chem. 2007 Nov 16;282(46):33444-33451
references.pmid: 17893144
references.citation: Genes Dev. 2005 Aug 15;19(16):1905-19
references.pmid: 16103218
references.citation: Nucleic Acids Res. 2005 Jul 1;33(Web Server issue):W299-302
references.pmid: 15980475
references.citation: Bioinformatics. 2008 Dec 1;24(23):2780-1
references.pmid: 18818215
references.citation: Proc Natl Acad Sci U S A. 2006 Nov 21;103(47):17696-700
references.pmid: 17062752
references.citation: Proc Natl Acad Sci U S A. 2001 Aug 28;98(18):10037-41
references.pmid: 11517324
references.citation: Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2210-21
references.pmid: 15572774
references.citation: Annu Rev Biochem. 2001;70:39-80
references.pmid: 11395402
references.citation: J Biol Chem. 1989 Feb 5;264(4):2189-94
references.pmid: 2644256
references.citation: Biochemistry. 2002 Apr 16;41(15):4891-900
references.pmid: 11939784