Probing the function of individual amino acid residues in the DNA binding site of the EcoRI restriction endonuclease by analysing the toxicity of genetically engineered mutants.
We have developed an assay that allows analysis of the activity of EcoRI restriction endonuclease (ENase) and its mutants in vivo. This assay is based on the fact that wild type (wt) EcoRI ENase is toxic for Escherichia coli cells not expressing the EcoRI methyltransferase (MTase). The viability factor defined by the ratio of the viable counts of E. coli cultures having or not having expressed the ecoRIR gene for a defined time is 10(-6) for wt EcoRI ENase and close to one for a totally inactive EcoRI ENase mutant. While the EcoRI MTase (M.EcoRI) provides substantial protection against the toxic effects of the wt EcoRI ENase and several of the mutants, some mutants become more toxic in the presence of M.EcoRI. Twenty-four different DNA-binding-site mutants of EcoRI ENase were characterized in their activity in vivo with this assay. The results obtained allow us to conclude that the structural integrity of the region at and around aa 200 seems to be very critical for the enzymatic function of EcoRI ENase: nonconservative replacements there lead to viability factors of 1-10(-2). While our results indicate that the region around aa 144 and 145 is also involved in the EcoRI ENase-catalyzed reaction, it is also evident that the effects of mutation there are not as large: viability factors of approx. 10(-3) are obtained even for drastic replacements. These results are discussed in the light of the x-ray structure analysis of an EcoRI ENase-DNA recognition complex.
Oelgeschläger
T
T
Zentrum Biochemie, Medizinische Hochschule Hannover, F.R.G.
Geiger
R
R
Rüter
T
T
Alves
J
J
Fliess
A
A
Pingoud
A
A
D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000235:genetics; D001665:Binding Sites; D004279:DNA, Viral / Q000378:metabolism; D015246:Deoxyribonuclease EcoRI / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D005822:Genetic Vectors; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011487:Protein Conformation; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D004279:DNA, Viral; C020098:DNA modification methylase EcoRI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D015246:Deoxyribonuclease EcoRI