Conversion of the tetrameric restriction endonuclease Bse634I into a dimer: oligomeric structure-stability-function correlations.
The Bse634I restriction endonuclease is a tetramer and belongs to the type IIF subtype of restriction enzymes. It requires two recognition sites for its optimal activity and cleaves plasmid DNA with two sites much faster than a single-site DNA. We show that disruption of the tetramerisation interface of Bse634I by site-directed mutagenesis converts the tetrameric enzyme into a dimer. Dimeric W228A mutant cleaves plasmid DNA containing one or two sites with the same efficiency as the tetramer cleaves the two-site plasmid. Hence, the catalytic activity of the Bse634I tetramer on a single-site DNA is down-regulated due to the cross-talking interactions between the individual dimers. The autoinhibition within the Bse634I tetramer is relieved by bridging two DNA copies into the synaptic complex that promotes fast and concerted cleavage at both sites. Cleavage analysis of the oligonucleotide attached to the solid support revealed that Bse634I is able to form catalytically competent synaptic complexes by bridging two molecules of the cognate DNA, cognate DNA-miscognate DNA and cognate DNA-product DNA. Taken together, our data demonstrate that a single W228A mutation converts a tetrameric type IIF restriction enzyme Bse634I into the orthodox dimeric type IIP restriction endonuclease. However, the stability of the dimer towards chemical denaturants, thermal inactivation and proteolytic degradation are compromised.
Zaremba
M
M
Institute of Biotechnology, Graiciuno 8, Vilnius LT-02241, Lithuania.
Sasnauskas
G
G
Urbanke
C
C
Siksnys
V
V
D002850:Chromatography, Gel; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004795:Enzyme Stability; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D010957:Plasmids / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D013696:Temperature; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism; D014461:Ultracentrifugation
D014364:Tryptophan; D004247:DNA; D004262:DNA Restriction Enzymes