Volume 5 Number 5 May 1978

Nucleic Acids Research

DNA-binding nonhistone proteins: DNA site reassociation

L. L. Jagodzinski, J.C. Chilton, and J.S. Sevall

Department of Chemistry, Texas Tech University, Lubbock, TX 79409, USA

Received 1 December 1977

ABSTRACT The DIRA-binding nonhistone proteins (UHF) have been demonstrated to fractionate the rat genome into protein-bound and unbound DNA sequences. Twenty percent of highly sheared rat DNA [X~350 base pair (bp)] can be retained on membrane filters as protein complexes. When extracted from the filter and retitrated with the NHP, a 4- to 5-fold enrichment of binding sites is present in the bound DNA with few, if any, sites detected in the unbound DNA. Rat DNA restricted by EcoRI endonuclease can be fractionated by its DNA-binding IH retention characteristics. Reassociation kinetics of the bound restricted sequences indicate that 45.6% is a subset of total single-copy sequence of the rat genome and 26.9% is repetitive sequences. Cross hybridization studies indicate the repetitive sequences of the bound DNAare not enriched as much as the slow component of the rat genome. Thus a 4-fold enrichment of a subset of the rat genome has been observed via NHP-DNA interactions. IlTRODUCTION There is accumulating evidence that one mechanism for differential gene expression is the sequence specific regulation of DNA transcription. A universal highly ordered pattern of repetitive sequence organization in the DNiA of a variety of eukaryotic organisms is well established (1-3). The proximity of subsets of repeated sequences to transcribed and translated sequences supports the functional role of this pattern in gene regulation (4,5). Seventy percent of the rat genome is characterized by a sequence pattern distribution with 200 to 400 nucleotide pair repetitive sequences interspersed among 2400 nucleotide pair singlecopy sequences (6). The possibility that the nonhistone proteins (NHP) of eukaryotic chromosomes have an active role in gene expression has emerged from experiments using chromatin reconstitution and DIA-R1qA hybridization (7-9). It has been suggested that NHP might interact specifically with histones or DNA (10-14). By their binding, the 11111P can induce a

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Nucleic Acids Research structural change in chromatin (15,16) or affect the thermal stability of the DNIA (17,18). Analysis of the rat liver INH-DIA interactions indicates that there is an apparent preference for rat DNA repetitive sequences (19). In the present work, we have investigated the sequence specificity in in vitro DNA-binding nonhistone protein-DlNA complexes. A group of rat liver DNA-binding NHP has been isolated that can retain high affinity DNA-binding sites as protein-DNA complexes. Reassociation kinetics of the retained DUIA indicate that a subset of the rat genome has been isolated with respect to the repetitive and single-copy D'A sequences. IATERIALS AND MET-HODS Protein and DN4A preparation: Nonhistone proteins were purified from rat liver nuclei (Lesser, personal communication) as described previously (19). In all steps, difluorylphosphate (0.1 mM) was added to inhibit protease activity (20). DNA-binding NHP were isolated by phosphocellulose chromatography which retained all NHP with DNA affinity as determined by a tandem rat DNA cellulose column which retained no proteins. Rat DNA was purified from crude chromatin by standard techniques (21,22) and mechanically sheared to 350 nucleotide pairs (30) or restricted with EcoRI (23). In vitro labeling of the DNA by the DNA polymerase I technique (24) or by the in vitro iodination method of Commerford (25-27) provided specific activities between 105 - 106 cpm/hg DNA. DNA reassociation was monitored by hydroxylapatite (HAP) chromatography (28) as described previously (29,30).

Isolation of bound DNA: Protein DNA-binding sites (BDNA) were separated from nonbinding sites (UBDNA) by filtering a protein-DNA mixture over 47 mm diameter,

0.45 p pore size Schleicher and Schuell Filters. Proteins and DNA were mixed in reaction buffer 0.04 M phosphate (pH 6.8), 1 X 104 M MgC2, 1 X 10 M EDTA, 0.1l B-mercaptoethanol, 0.2 M NaCl, and 5 pg/ml bovine serum albumin at mass rations of 4-6 pg:l pg DNA, respectively (19). No more than 0.5 mg of protein was filtered over any one filter. The filter was washed with buffer and BDNA eluted twice with 2 ml of 0.1% sodium godecyl sulfate (SDS), followed by 1 ml of buffer rinse. 1488

Nucleic Acids Research RESSULTS

Isolation of protein-bound DNA: Interaction between protein and DNA can be investigated by the retention of protein and any associated DNA on membrane filters (43,44). Through the use of SDS treatment (31), it is possible to release the DINA in protein DNA complexes on membrane filters. Under "stringent" binding conditions [0.20 NaCl, 0.04 M phosphate (p4, 6.8)], 20-25% of 350 nucleotide rat DNA can be retained by saturating amounts of DNA-binding l1P (19). Greater than 95% of the bound DNA could be removed with 0.1% SDS (1.5ml/cm2). The detergent also solubilized much of the membrane filter requiring extensive phenol extraction of the DN4A solution. The bound and unbound 350 nucleotide DNA fragments were purified and retitrated with the DNA-binding rat liver NHP at 0.20 M iiaCl, 0.04 M phosphate (pH 6.8)(Figure 1). Greater than 50% of the bound DNA was retained at mass ratios of protein to DNIA of 40:1; whereas 10% of the -

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DNA-binding nonhistone proteins: DNA site reassociation.

Volume 5 Number 5 May 1978 Nucleic Acids Research DNA-binding nonhistone proteins: DNA site reassociation L. L. Jagodzinski, J.C. Chilton, and J.S...
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