A complex composed of at least two HeLa nuclear proteins protects preferentially one DNA strand of the simple (gt)n(ga)m containing region of intron 2 in HLA-DRB genes.
Electrophoretic mobility shift assays reveal that HeLa nuclear proteins bind fast and with measurable affinity to target DNAs containing mixed simple repetitive (gt)n(ga)m stretches. Preincubation of the proteins at elevated temperature prevents the formation of the major DNA/protein complex in favour of several distinct assemblies. A similar pattern of retarded bands was observed employing higher salt concentrations in the binding reaction. Thus conformational changes of different proteins appear to influence the complex rather than alternating DNA structures. Separation of the total nuclear extract into a water soluble and an insoluble protein fraction leads to a complete loss of target DNA binding capability of the fractions. The binding capacity is restored by combining the two fractions suggesting that at least two protein components are necessary to form a complex with the target sequence. The proteins can be differentiated into heat sensitive, water soluble and temperature stable, water insoluble, respectively. Furthermore, specifically binding polypeptides are not detectable by Southwestern analyses, probably because the essential components are separated during electrophoresis. DNase I footprint analyses yield four different protein binding regions only on the (gt)n(ga)m harbouring strand. The footprints cover larger portions of the mixed simple repeat in addition to a portion 5' of the (gt)n part. Hence at least two nuclear protein components of unknown biological function have to be present simultaneously to protect preferentially the (gt)n(ga)m-containing strand of intron 2 in HLA-DRB genes.
Mäueler
W
W
Molekulare Humangenetik, Ruhr-Universität, Bochum, Germany.
Frank
G
G
Muller
M
M
Epplen
J T
JT
D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D003850:Deoxyribonuclease I; D006684:HLA-DR Antigens / Q000235:genetics*; D059811:HLA-DRB1 Chains; D006367:HeLa Cells; D006358:Hot Temperature; D006801:Humans; D015151:Immunoblotting; D007438:Introns; D007700:Kinetics; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid
D006684:HLA-DR Antigens; D059811:HLA-DRB1 Chains; D009687:Nuclear Proteins; D004247:DNA; D003850:Deoxyribonuclease I