The (gt)n(ga)m containing intron 2 of HLA-DRB alleles binds a zinc-dependent protein and forms non B-DNA structures.
We studied protein binding and structural features of perfect and imperfect composite (gt)n(ga)m blocks from different HLA-DRB1 alleles in their original genomic and artificial environments. The major retarded protein/DNA complex of the genomic (gt)n(ga)m fragments comprises a zinc-dependent protein present in nuclear extracts from different cell types. The protein binding is characterized by moderate affinities independent of the polymorphic form of the physiological microsatellite allele. The binding affinity depends on the 5' and 3' adjacent single copy parts. DNase I footprinting of genome-derived fragments revealed that the 5' adjacent sequence and the (gt)n repeat are preferentially protected on the (gt)n(ga)m strand. Comparing three alleles, a regular pattern of footprints was not detectable in the (gt)n part, indicating that the zinc-dependent protein recognizes structural rather than sequence-specific features in this region. Chemical probing resulted in a pattern characteristic for Z-DNA in the (gt)n tract of the fragments. However, EMSA experiments using the Z-DNA specific monoclonal antibody mABZ-22 did not prove the presence of Z-DNA. As demonstrated by chemical modifications of the different (ga)m targets, only one of three (gt)n(ga)m fragments formed intramolecular triplexes of the type H-y3 and H-y5. DNase I footprinting revealed only weak protection, if any, in the homopurine tract. Rather, the (tc)m strands are hypersensitive for DNase I. This is probably due to structural conversions into intramolecular *H-triplexes after binding of HIZP.
Mäueler
W
W
Molekulare Humangenetik, Ruhr-Universität, 44801, Bochum, Germany.
Bassili
G
G
Arnold
R
R
Renkawitz
R
R
Epplen
J T
JT
D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D004247:DNA / Q000737:chemistry; D006684:HLA-DR Antigens / Q000235:genetics* / Q000378:metabolism*; D059811:HLA-DRB1 Chains; D006367:HeLa Cells / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007438:Introns; D008214:Lymphocytes / Q000378:metabolism; D018895:Microsatellite Repeats / Q000235:genetics*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides; D015032:Zinc / Q000378:metabolism
D006684:HLA-DR Antigens; D059811:HLA-DRB1 Chains; D009687:Nuclear Proteins; D009841:Oligonucleotides; D004247:DNA; D015032:Zinc