GATA-1 bends DNA in a site-independent fashion

J Biol Chem. 2000 Sep 8;275(36):28152-6. doi: 10.1074/jbc.M002053200.

Abstract

The DNA binding domain of GATA-1 consists of two adjacent homologous zinc fingers, of which only the C-terminal finger binds DNA independently. Solution structure studies have shown that the DNA is bent by about 15 degrees in the complex formed with the single C-terminal finger of GATA-1. The N-terminal finger stabilizes DNA binding at some sites. To determine whether it contributes to DNA bending, we have performed circular permutation DNA bending experiments with a variety of DNA-binding sites recognized by GATA-1. By using a series of full-length GATA-1, double zinc finger, and single C-terminal finger constructs, we show that GATA-1 bends DNA by about 24 degrees, irrespective of the DNA-binding site. We propose that the N- and C-terminal fingers of GATA-1 adopt different orientations when bound to different cognate DNA sites. Furthermore, we characterize circular permutation bending artifacts arising from the reduced gel mobility of the protein-DNA complexes.

MeSH terms

  • Animals
  • Base Sequence
  • Binding Sites
  • Carrier Proteins
  • Chickens
  • DNA / chemistry*
  • DNA / metabolism
  • DNA-Binding Proteins / metabolism*
  • Erythroid-Specific DNA-Binding Factors
  • GATA1 Transcription Factor
  • Glutathione Transferase
  • Humans
  • Maltose-Binding Proteins
  • Molecular Sequence Data
  • Nuclear Proteins / metabolism
  • Nucleic Acid Conformation*
  • Oligodeoxyribonucleotides / chemistry
  • Plasmids / chemistry*
  • Plasmids / metabolism
  • Recombinant Fusion Proteins / metabolism
  • Restriction Mapping
  • Transcription Factors / metabolism*
  • Zinc Fingers

Substances

  • Carrier Proteins
  • DNA-Binding Proteins
  • Erythroid-Specific DNA-Binding Factors
  • GATA1 Transcription Factor
  • GATA1 protein, human
  • Maltose-Binding Proteins
  • Nuclear Proteins
  • Oligodeoxyribonucleotides
  • Recombinant Fusion Proteins
  • Transcription Factors
  • DNA
  • Glutathione Transferase