title: Crystallization and preliminary X-ray analysis of a DNA primase from hyperthermophilic archaeon Pyrococcus horikoshii.
abstract: At the initiation of chromosomal DNA replication, DNA primases synthesize short RNA primers, which are subsequently elongated by DNA polymerases. To understand the structural basis for the primer synthesis by archaeal/eukaryotic-type primases, the gene of the DNA primase from hyperthermophilic archaeon Pyrococcus horikoshii was cloned and overexpressed in Escherichia coli as a fusion protein with a hexa-histidine tag at its amino terminus. The recombinant DNA primase was purified and crystallized by the hanging-drop vapor diffusion method at 293 K, with polyethylene glycol 8000 as the precipitant. The crystals belong to the P3(2)21 space group with unit-cell parameters a = b = 77.8, c = 129.6 A, and alpha = beta = 90 degrees, gamma = 120 degrees. Crystals of the selenomethionine derivative were obtained by means of a cross-seeding method using native crystals. The data for the native and selenomethionine-substituted crystals were collected to 1.8 and 2.2 A resolution, respectively, with synchrotron radiation at SPring-8 under flash-frozen conditions at 100 K. The four wavelength MAD data provided a phase to determine the structure of the primase at 2.2 A resolution.
authors.affiliation: Cellular Physiology Laboratory, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
authors.forename: N
authors.initials: N
authors.lastname: Ito
authors.forename: O
authors.initials: O
authors.lastname: Nureki
authors.forename: M
authors.initials: M
authors.lastname: Shirouzu
authors.forename: S
authors.initials: S
authors.lastname: Yokoyama
authors.forename: F
authors.initials: F
authors.lastname: Hanaoka
mesh_terms: D019943:Amino Acid Substitution; D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods*; D019915:DNA Primase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011487:Protein Conformation; D019711:Pyrococcus / Q000201:enzymology*; D012313:RNA / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012645:Selenomethionine / Q000737:chemistry
chemical_list: C034477:RNA primers; D011994:Recombinant Proteins; D012313:RNA; D012645:Selenomethionine; D019915:DNA Primase