Crystallization and preliminary X-ray analysis of a DNA primase from hyperthermophilic archaeon Pyrococcus horikoshii.
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.
Ito
N
N
Cellular Physiology Laboratory, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
Nureki
O
O
Shirouzu
M
M
Yokoyama
S
S
Hanaoka
F
F
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
C034477:RNA primers; D011994:Recombinant Proteins; D012313:RNA; D012645:Selenomethionine; D019915:DNA Primase