Molecular cloning of the three base restriction endonuclease R.CviJI from eukaryotic Chlorella virus IL-3A.
R.CviJI is unique among site-specific restriction endonucleases in that its activity can be modulated to recognize either a two or three base sequence. Normally R.CviJI cleaves RGCY sites between the G and C to leave blunt ends. In the presence of ATP R.CviJI* cleaves RGCN and YGCY sites, but not YGCR sites. The gene encoding R.CviJI was cloned from the eukaryotic Chlorella virus IL-3A and expressed in Escherichia coli. The primary E.coli cviJIR gene product is a 278 amino acid protein initiated from a GTG codon, rather than the expected 358 amino acid protein initiated from an in-frame upstream ATG codon. Interestingly, the 278 amino acid protein displays the normal restriction activity but not the R.CviJI* activity of the native enzyme. Nine restriction and modification proteins which recognize a central GC or CG sequence share short regions of identity with R.CviJI amino acids 144-235, suggesting that this region is the recognition and/or catalytic domain.
Swaminathan
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N
CHIMERx, Madison, WI 53704, USA.
Mead
D A
DA
McMaster
K
K
George
D
D
Van Etten
J L
JL
Skowron
P M
PM
D000595:Amino Acid Sequence; D001483:Base Sequence; D002708:Chlorella / Q000821:virology*; D003001:Cloning, Molecular; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D005057:Eukaryotic Cells / Q000821:virology; D005814:Genes, Viral; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017862:Phycodnaviridae / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000302:isolation & purification; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid
D011994:Recombinant Proteins; C075913:endodeoxyribonuclease CviJI; D015252:Deoxyribonucleases, Type II Site-Specific