Expression, purification, crystallization and preliminary crystallographic analysis of a thermostable DNA ligase from the archaeon Thermococcus sibiricus.
DNA ligases join single-strand breaks in double-stranded DNA by catalyzing the formation of a phosphodiester bond between adjacent 5'-phosphate and 3'-hydroxyl termini. Their function is essential to maintain the integrity of the genome in DNA replication, recombination and repair. A recombinant ATP-dependent DNA ligase from the hyperthermophilic anaerobic archaeon Thermococcus sibiricus was expressed in Escherichia coli and purified. Crystals were grown by vapour diffusion using the hanging-drop method with 17%(w/v) PEG 4000 and 8.5%(v/v) 2-propanol as precipitants. A diffraction experiment was performed with a single crystal, which diffracted X-rays to 3.0 Å resolution. The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 58.590, b = 87.540, c = 126.300 Å.
Bach Institute of Biochemistry, RAS, Moscow, Russian Federation. petrova@impb.psn.ru
T E
TE
Petrova
E Y
EY
Bezsudnova
B D
BD
Dorokhov
E S
ES
Slutskaya
K M
KM
Polyakov
P V
PV
Dorovatovskiy
N V
NV
Ravin
K G
KG
Skryabin
M V
MV
Kovalchuk
V O
VO
Popov
D003460:Crystallization; D018360:Crystallography, X-Ray; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004795:Enzyme Stability; D015870:Gene Expression; D013696:Temperature; D019712:Thermococcus / Q000201:enzymology*
D011088:DNA Ligases; D000072481:DNA Ligase ATP