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

Abstract

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.

Genomic simple repetitive DNAs are targets for differential binding of nuclear proteins

“…The protein binding to a double-stranded (GAA)24 block (Fig. 1, right-hand side) is characterized by high affinity binding comparable to that measured in experiments involving dinucleotide repeats (see below and [29]). DNA footprinting results show that only the (GAA)n repeat of the polypurine strand is preferentially protected by the bound protein against DNase I digestion, whereas the complement is not.…”

“…As a prerequisite for specific protein binding to (GT), repeats there must be a minimum length. Nuclear proteins do not bind to (GT)n motifs of 6 dinucleotide units whereas 13-mers are bound [29]. This conclusion is supported by the observation that double-stranded (GT)s/(AC)8 oligonucleotides cannot compete for binding [29].…”

“…Clearly, the binding characteristics for different motifs are different. Different targets bind different nuclear proteins [25][26][27][28][29][30] (Table 1). The binding affinities are not only based on the motifs, but they also depend on a minimal length of the simple repeat block (see Fig.…”

“…-+++ aFor all methodological details and further explanations see [25][26][27][28][29]. bEach of the simple repeat targets was an efficient competitor for protein binding whenever itself was the labelled target.…”

“…Nuclear proteins do not bind to (GT)n motifs of 6 dinucleotide units whereas 13-mers are bound [29]. This conclusion is supported by the observation that double-stranded (GT)s/(AC)8 oligonucleotides cannot compete for binding [29]. Footprint analyses revealed that, in (GT), blocks, similarly to the mixed (GT)n(GA)m (see below and [29]), repeatadjacent, non-tandem sequences are obligatorily protected from DNase I digestion (M~iueler et al, in preparation).…”