An EcoRI-RsrI chimeric restriction endonuclease retains parental sequence specificity

Tungalag Chuluunbaatar, Tetiana Ivanenko-Johnston, M. Fuxreiter, Ruslan Meleshko, Tamás Raskó, I. Simon, Joseph Heitman, A. Kiss

Research output: Contribution to journalArticle

3 Citations (Scopus)

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 ∼ 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.

Original languageEnglish
Pages (from-to)583-594
Number of pages12
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Volume1774
Issue number5
DOIs
Publication statusPublished - May 2007

Fingerprint

Deoxyribonuclease EcoRI
Endonucleases
DNA Restriction Enzymes
Poisons
Amino Acids
Molecular modeling
DNA sequences
Methyltransferases
Molecular dynamics
Inclusion Bodies
Hydrogen bonds
Substitution reactions
Amino Acid Substitution
Molecular Dynamics Simulation
Hydrogen
Amino Acid Sequence
DNA
Computer simulation
Substrates
Enzymes

Keywords

  • Molecular dynamics simulations
  • Protein refolding
  • Restriction endonuclease
  • Sequence-specific DNA - recognition

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Genetics

Cite this

An EcoRI-RsrI chimeric restriction endonuclease retains parental sequence specificity. / Chuluunbaatar, Tungalag; Ivanenko-Johnston, Tetiana; Fuxreiter, M.; Meleshko, Ruslan; Raskó, Tamás; Simon, I.; Heitman, Joseph; Kiss, A.

In: Biochimica et Biophysica Acta - Proteins and Proteomics, Vol. 1774, No. 5, 05.2007, p. 583-594.

Research output: Contribution to journalArticle

Chuluunbaatar, Tungalag ; Ivanenko-Johnston, Tetiana ; Fuxreiter, M. ; Meleshko, Ruslan ; Raskó, Tamás ; Simon, I. ; Heitman, Joseph ; Kiss, A. / An EcoRI-RsrI chimeric restriction endonuclease retains parental sequence specificity. In: Biochimica et Biophysica Acta - Proteins and Proteomics. 2007 ; Vol. 1774, No. 5. pp. 583-594.
@article{db2f5df80426435199ddf0285e3812d6,
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 ∼ 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.",
keywords = "Molecular dynamics simulations, Protein refolding, Restriction endonuclease, Sequence-specific DNA - recognition",
author = "Tungalag Chuluunbaatar and Tetiana Ivanenko-Johnston and M. Fuxreiter and Ruslan Meleshko and Tam{\'a}s Rask{\'o} and I. Simon and Joseph Heitman and A. Kiss",
year = "2007",
month = "5",
doi = "10.1016/j.bbapap.2007.02.011",
language = "English",
volume = "1774",
pages = "583--594",
journal = "Biochimica et Biophysica Acta - Proteins and Proteomics",
issn = "1570-9639",
publisher = "Elsevier",
number = "5",

}

TY - JOUR

T1 - An EcoRI-RsrI chimeric restriction endonuclease retains parental sequence specificity

AU - Chuluunbaatar, Tungalag

AU - Ivanenko-Johnston, Tetiana

AU - Fuxreiter, M.

AU - Meleshko, Ruslan

AU - Raskó, Tamás

AU - Simon, I.

AU - Heitman, Joseph

AU - Kiss, A.

PY - 2007/5

Y1 - 2007/5

N2 - 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 ∼ 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.

AB - 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 ∼ 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.

KW - Molecular dynamics simulations

KW - Protein refolding

KW - Restriction endonuclease

KW - Sequence-specific DNA - recognition

UR - http://www.scopus.com/inward/record.url?scp=34247582888&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=34247582888&partnerID=8YFLogxK

U2 - 10.1016/j.bbapap.2007.02.011

DO - 10.1016/j.bbapap.2007.02.011

M3 - Article

C2 - 17442645

AN - SCOPUS:34247582888

VL - 1774

SP - 583

EP - 594

JO - Biochimica et Biophysica Acta - Proteins and Proteomics

JF - Biochimica et Biophysica Acta - Proteins and Proteomics

SN - 1570-9639

IS - 5

ER -