title: An EcoRI-RsrI chimeric restriction endonuclease retains parental sequence specificity.
abstract: To test their structural and functional similarity, hybrids were constructed between EcoRI and RsrI, two restriction endonucleases recognizing the same DNA sequence and sharing 50% amino acid sequence identity. One of the chimeric proteins (EERE), in which the EcoRI segment His147-Ala206 was replaced with the corresponding RsrI segment, showed EcoRI/RsrI-specific endonuclease activity. EERE purified from inclusion bodies was found to have approximately 100-fold weaker activity but higher specific DNA binding affinity, than EcoRI. Increased binding is consistent with results of molecular dynamics simulations, which indicate that the number of hydrogen bonds formed with the recognition sequence increased in the chimera as compared to EcoRI. The success of obtaining an EcoRI-RsrI hybrid endonuclease, which differs from EcoRI by 22 RsrI-specific amino acid substitutions and still preserves canonical cleavage specificity, is a sign of structural and functional similarity shared by the parental enzymes. This conclusion is also supported by computational studies, which indicate that construction of the EERE chimera did not induce substantial changes in the structure of EcoRI. Surprisingly, the chimeric endonuclease was more toxic to cells not protected by EcoRI methyltransferase, than the parental EcoRI mutant. Molecular modelling revealed structural alterations, which are likely to impede coupling between substrate recognition and cleavage and suggest a possible explanation for the toxic phenotype.
authors: Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, P.O. Box 521, 6701 Szeged, Hungary.
authors: Tungalag
authors: T
authors: Chuluunbaatar
authors: Tetiana
authors: T
authors: Ivanenko-Johnston
authors: Mónika
authors: M
authors: Fuxreiter
authors: Ruslan
authors: R
authors: Meleshko
authors: Tamás
authors: T
authors: Raskó
authors: István
authors: I
authors: Simon
authors: Joseph
authors: J
authors: Heitman
authors: Antal
authors: A
authors: Kiss
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D015246:Deoxyribonuclease EcoRI / Q000737:chemistry / Q000378:metabolism*; D006860:Hydrogen Bonding; D008958:Models, Molecular; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D011993:Recombinant Fusion Proteins; C020098:DNA modification methylase EcoRI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D015246:Deoxyribonuclease EcoRI