Intra- vs intersubunit communication in the homodimeric restriction enzyme EcoRV: Thr 37 and Lys 38 involved in indirect readout are only important for the catalytic activity of their own subunit

Biochemistry
F StahlA Pingoud

Abstract

EcoRV is a dimer of two identical subunits which together form one binding site for the double-stranded DNA substrate. Concerted cleavage of both strands of the duplex requires intersubunit communication to synchronize the two catalytic centers of EcoRV. Here we address the question of how contacts to the DNA backbone trigger conformational changes which lead to the activation of both catalytic centers. The structure of the specific EcoRV-DNA complex shows that a region including amino acids Thr 37 and Lys 38 is involved in interactions with the DNA backbone and is a candidate for intersubunit communication. Homodimeric EcoRV T37A and K38A variants have a 1000-fold reduced catalytic activity. To examine whether Thr 37 and Lys 38 of one subunit affect the catalytic center in the same subunit and/or in the other subunit, we have produced heterodimeric variants containing a Thr 37 --> Ala or Lys 38 --> Ala substitution in one subunit combined with a wild type (wt) subunit (wt/T37A and wt/K38A) or with a subunit which contains an amino acid substitution (Asp 90 --> Ala) in the active site (D90A/T37A and D90A/K38A). Cleavage experiments with supercoiled pAT153 show that wt/T37A and wt/K38A preferentially nick the DNA. A steady-state...Continue Reading

References

Oct 1, 1993·European Journal of Biochemistry·V Siksnys, M Pleckaityte
May 15, 1997·European Journal of Biochemistry·A Pingoud, A Jeltsch

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Citations

Nov 26, 2002·Journal of Molecular Biology·Christine RauchKlaus R Liedl
Sep 15, 2001·Nucleic Acids Research·A Pingoud, A Jeltsch
Jul 13, 2004·Nucleic Acids Research·James C SamuelsonShuang-yong Xu
Mar 10, 2010·Biochemistry. Biokhimii︠a︡·L A ZheleznayaN I Matvienko
Aug 26, 2010·Biochemistry·Kommireddy VasuValakunja Nagaraja

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