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ABSTRACTa-Protein is a high mobility group protein originally purified from African green monkey cells based on its affinity for the 172-base-pair repeat of monkey a-satellite DNA. We have used DNase I footprinting to identify 50 a-protein binding sites on simian virus 40 DNA and thereby to determine the DNA binding specificity of this mammalian nuclear protein. a-Protein binds with approximately equal affinity to any run of six or more APT base pairs in duplex DNA, to many, if not all, runs of five AkT base pairs, and to a small number of other sequences within otherwise (A+T)-rich regions. Unlike well characterized sequence-specific DNA binding proteins such as bacterial repressors, a-protein makes extensive contacts within the minor groove of B-DNA. These and related findings indicate that, rather than binding to a few specific DNA sequences, a-protein recognizes a configuration of the minor groove characteristic of short runs of AT base pairs. We discuss possible functions of a-protein and the similarities in DNA recognition by a-protein and the antibiotic netropsin.Previous studies from this laboratory have addressed the existence and properties of DNA sequence-specific nucleosome-binding proteins (1-3). In particular, we searched for a protein specific for the a-satellite DNA (a-DNA) of the African green monkey. Using the "band-competition" assay, a generally applicable electrophoretic assay for specific DNA-binding proteins in crude extracts, we purified an abundant nuclear protein from green monkey CV-1 cells that preferentially bound to a-DNA (1). The solubility properties, amino acid composition, and primary structure of this =10 kDa protein (tentatively called a-protein) operationally classified it as a high mobility group (HMG) protein (4-6), distinct from the other major HMG proteins, HMG 1, -2, -14, and -17 (J. McCartney, F.S., M.J.S., J. Smart, and A.V., unpublished data). The preferred a-nucleosome frame detected in isolated chromatin (7,8) is precisely bordered by a-protein binding sites (GATAT'IT) on a-DNA, suggesting that aprotein might function as a nucleosome-positioning or phasing protein (1).To address the binding specificity of a-protein in more detail, we mapped a-protein binding sites on simian virus 40 (SV40) DNA. a-Protein binds with approximately equal affinity not only to the GATATTT sequences in SV40 DNA but also to >50 other sites in the -2.4 kilobase pairs (kbp) that we have examined by DNase I footprinting. Thus, rather than recognizing a few specific nucleotide sequences, aprotein recognizes an aspect of B-DNA conformations, most likely a configuration of the minor groove, that is characteristic of short runs of APT base pairs.These and other properties of a-protein set it apart from the more extensively 'studied prokaryotic and eukaryotic sequence-specific DNA binding proteins, whose characteristic features include the predominance of major groove interactions and little or no sequence degeneracy in DNA recognition (9). MATERIALS AND METHODSDNase I Footprinting. DNA fragments ...

Ku protein is a relatively abundant DNA-binding protein which was first detected as the autoantigen in a patient with scleroderma-polymyositis overlap syndrome (hence the name 'Ku'). It is a heterodimer of two polypeptide chains of molecular weights 85,000 and 72,000, and it characteristically binds, in vitro, to the ends of DNA fragments, and translocates to form regular multimeric complexes, with one protein bound per 30 bp of DNA. We have studied the mechanism of interaction of Ku protein with DNA in vitro, using protein extracted from cultured monkey cells. We find that the precise structure of the DNA ends is not important for binding, as Ku protein can bind to hairpin loops and to mononucleosomes. Bound protein also does not require DNA ends for continued binding, since complexes formed with linear DNAs can be circularized by DNA ligase. Dissociation of the complex also appears to require DNA ends, since ligase closed circular complexes were found to be extremely stable even in the presence of 2 M NaCl. We also found that Ku molecules slide along DNA, with no preferential binding to specific sequences. Thus, Ku protein behaves like a bead threaded on a DNA string, a binding mechanism which allows us to make a new hypothesis concerning the function of this protein in the nucleus.

Figure 1 shows that precipitating picogram amounts of DNA by the above method in the presence of polyacrylamide results in complete recovery of fragments larger than 20 base pairs, whereas most of the DNA is lost if no carrier is used. Also note that very short DNA fragments do not coprecipitate with polyacrylamide, which permits separation of labeled DNA from unreacted nucleotides by precipitation after labeling reactions. Polyacrylamide has been used in several laboratories for most of the common manipulations of DNA, including enzyme reactions, gel electrophoresis, cloning (3), DNA-protein interactions (2), and appears inert in all experiments. For example, unlike tRNA, it is not phosphorylated with polynucleotide kinase, and Figure 2 shows an example where glycogen interferes with DNA-protein interactions whereas polyacrylamide does not.

The tandemly repeated DNA sequence poly(CA).poly(TG) is found in tracts up to 60 base pairs long, dispersed at thousands of sites throughout the genomes of eukaryotes. Double-stranded DNA fragments containing such sequences associated spontaneously with each other in vitro, in the absence of protein, forming stable four-stranded structures that were detected by gel electrophoresis and electron microscopy. These structures were recognized specifically by the nuclear nonhistone high mobility group (HMG) proteins 1 and 2 as evidenced by gel retardation. Such sequence-specific complexes might be involved in vivo in recombination or other processes requiring specific association of two double-stranded DNA molecules.