Distance determination by GIY-YIG intron endonucleases: discrimination between repression and cleavage functions

Nucleic Acids Res. 2006 Mar 31;34(6):1755-64. doi: 10.1093/nar/gkl079. Print 2006.

Abstract

GIY-YIG homing endonucleases are modular proteins, with conserved N-terminal catalytic domains connected by linkers to C-terminal DNA-binding domains. I-TevI, the T4 phage GIY-YIG intron endonuclease, functions both in promoting td intron homing, and in acting as a transcriptional autorepressor. Repression is achieved by binding to an operator, which is cleaved at 100-fold reduced efficiency relative to the intronless homing site. The linker includes a zinc finger, which functions in distance determination, to constrain the catalytic domain to cleave the homing site at a fixed position. Here we show that I-BmoI, a related GIY-YIG endonuclease lacking a zinc finger, also possesses some cleavage distance discrimination. Furthermore, hybrid endonucleases constructed by swapping the domains of I-BmoI and I-TevI are active, precise and demonstrate that features other than the zinc finger facilitate distance determination. Most importantly, I-TevI zinc finger mutants cleave the operator more efficiently than the homing site, the converse of wild-type protein. These results are consistent with the zinc finger acting as a measuring device, directing efficient cleavage of the homing site to promote intron mobility, while reducing cleavage at the operator to ensure transcriptional autorepression and phage viability.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Binding Sites
  • Catalytic Domain
  • DNA / chemistry
  • DNA / metabolism
  • Endodeoxyribonucleases / chemistry*
  • Endodeoxyribonucleases / genetics
  • Endodeoxyribonucleases / metabolism*
  • Introns*
  • Molecular Sequence Data
  • Mutation
  • Operator Regions, Genetic
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins / metabolism
  • Repressor Proteins / chemistry
  • Repressor Proteins / genetics
  • Repressor Proteins / metabolism
  • Substrate Specificity
  • Zinc Fingers

Substances

  • Recombinant Fusion Proteins
  • Repressor Proteins
  • DNA
  • Endodeoxyribonucleases
  • I-BmoI endonuclease
  • I-TEVI endonuclease