Crystallization and preliminary X-ray diffraction studies of Hje, a HolliDay junction resolving enzyme from Sulfolobus solfataricus.
HolliDay junction endonuclease (Hje) from Sulfolobus solfataricus is a resolving enzyme involved in cleaving specific sites on either side of recombinant four-way HolliDay junctions. The HJE gene from S. solfataricus was cloned from genomic DNA into the pET19b Escherichia coli expression vector and recombinant protein was expressed to high levels. Hje was purified using heat treatment, cation exchange and gel filtration. Hanging-drop crystallization trials yielded primitive hexagonal crystals which diffract to 2.4 A on a laboratory source. Systematic absences (only 00l = 6n present) and poor scaling in P622 indicate that the space group is P6(1) or its enantiomer. Failed attempts at molecular replacement using models of a related archaeal resolving enzyme, Hjc, raise the possibility of a difference in quaternary structure between Hjc and Hje, which may be responsible for differences in their activities.
Middleton
Claire L
CL
Division of Biological Chemistry and Molecular Microbiology, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland.
Parker
Joanne L
JL
Richard
Derek J
DJ
White
Malcolm F
MF
Bond
Charles S
CS
D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D017931:DNA Primers / Q000235:genetics; D019641:DNA, Archaeal / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D045565:Holliday Junction Resolvases; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D016944:Sulfolobus / Q000201:enzymology*
D017931:DNA Primers; D019641:DNA, Archaeal; D011994:Recombinant Proteins; D004706:Endodeoxyribonucleases; D045565:Holliday Junction Resolvases