A complex containing at least one zinc dependent HeLa nuclear protein binds to the intronic (gaa)(n) block of the frataxin gene.
We analyzed HeLa nuclear proteins binding to the (gaa)(n) harbouring intron 1 of nine frataxin alleles and characterized the structures of the repeats. Fragments with blocks longer than (gaa)(9) form spontaneously different intramolecular H-y topoisomeres in linear state. The observed triplexes depend on the length of the repeat. Interruption of the perfectly repeated (gaa)(n) block entails two structural regions. At least two HeLa nuclear proteins bind to the (gaa)(n) fragments resulting in a distinct major retarded complex as revealed by EMSA. One of these proteins is zinc dependent. Importantly, the fragment harbouring (gan)(121) binds additional proteins. Protein binding appears to be locus specific, and the binding affinity was found to be not random. The affinities of the different target fragments varied by a factor of four. Binding affinities of the fragments were not obviously correlated to differences in the composition of the repeats. DNase I footprinting revealed only weakly protected binding regions, but multiple HS sites in the repeat regions of the fragments. These findings and the fact, that DNA conformers observed in EMSA and electron microscopical experiments bind proteins, lead to the assumption that the proteins recognize, both, B-DNA and triple helical structures, but with different affinity. Possible functions of the proteins are discussed in the context of transformation of triple helical structures into B-DNA and the pathogenesis of FRDA.
Mäueler
W
W
Department of Molecular Human Genetics, Ruhr-Universität, 44780, Bochum, Germany. joerg.t.epplen@ruhr-uni-bochum.de
Bassili
G
G
Hardt
C
C
Keyl
H G
HG
Epplen
J T
JT
D000483:Alleles; D001483:Base Sequence; D001665:Binding Sites; D002413:Cations, Divalent / Q000494:pharmacology; D004247:DNA / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007438:Introns / Q000235:genetics; D033862:Iron-Binding Proteins; D008854:Microscopy, Electron; D009687:Nuclear Proteins / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D012689:Sequence Homology, Nucleic Acid; D018911:Trinucleotide Repeats; D015032:Zinc / Q000494:pharmacology*; D000096923:Frataxin
D002413:Cations, Divalent; D004247:DNA; D004268:DNA-Binding Proteins; D033862:Iron-Binding Proteins; D009687:Nuclear Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); D015032:Zinc; D000096923:Frataxin