The importance of the N-terminus of T7 endonuclease I in the interaction with DNA junctions

J Mol Biol. 2013 Jan 23;425(2):395-410. doi: 10.1016/j.jmb.2012.11.029. Epub 2012 Dec 1.

Abstract

T7 endonuclease I is a dimeric nuclease that is selective for four-way DNA junctions. Previous crystallographic studies have found that the N-terminal 16 amino acids are not visible, neither in the presence nor in the absence of DNA. We have now investigated the effect of deleting the N-terminus completely or partially. N-terminal deleted enzyme binds more tightly to DNA junctions but cleaves them more slowly. While deletion of the N-terminus does not measurably affect the global structure of the complex, the presence of the peptide is required to generate a local opening at the center of the DNA junction that is observed by 2-aminopurine fluorescence. Complete deletion of the peptide leads to a cleavage rate that is 3 orders of magnitude slower and an activation enthalpy that is 3-fold higher, suggesting that the most important interaction of the peptide is with the reaction transition state. Taken together, these data point to an important role of the N-terminus in generating a central opening of the junction that is required for the cleavage reaction to proceed properly. In the absence of this, we find that a cruciform junction is no longer subject to bilateral cleavage, but instead, just one strand is cleaved. Thus, the N-terminus is required for a productive resolution of the junction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacteriophage T7 / enzymology*
  • Binding Sites
  • DNA Primers / chemistry
  • DNA, Bacterial / chemistry
  • DNA, Bacterial / metabolism*
  • Deoxyribonuclease I / chemistry
  • Deoxyribonuclease I / genetics
  • Deoxyribonuclease I / metabolism*
  • Models, Molecular
  • Nucleic Acid Conformation
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism*
  • Protein Binding

Substances

  • DNA Primers
  • DNA, Bacterial
  • Peptide Fragments
  • Deoxyribonuclease I