DNA unwinding component of the nonhistone chromatin proteins.

Abstract

A subclass of nonhistone chromatin proteins from rat liver, previously shown to exhibit high affinity for DNA, has been fractionated by single-stranded DNA-agarose affinity chromatography. The protein fraction that bound to DNAagarose in 0.19 M NaCl-buffer and was eluted with 2 M NaClbuffer is enriched for a protein component of approximately 20,000 daltons and exhibits preferential binding to denatured DNA. This nonhistone protein fraction specific for single strands binds to DNA in a non-species-specific manner, and causes helix-coil transition of synthetic poly~d(A-T)d(A-T)J at 25°, as indicated by the increase in absorbance of ultraviolet light at 260 nm. The observed hyperchromicity does not result from any nuclease activity in the protein fraction, because addition of Mg+2 results in partial hypochromic shift, and the protein/DNA complex is retained by nitrocellulose filters. Protein/DNA interactions are clearly important in the structure and function of the eukaryotic chromatin. Interactions of histones with DNA have been extensively analyzed and, in recent years, have been shown to give rise to the subunit structure of the chromatin (1-5). The role of the nonhistone chromatin proteins (NHCP) in the chromatin is also a subject of much current interest. However, the NHCP are a heterogeneous class of proteins that have long resisted fractionation and characterization. Recently, the DNA binding properties of the NHCP have been exploited to fractionate and characterize them, and to gain insight into their possible biological roles (6-17).In our laboratory, we have isolated a distinct subclass of the NHCP, designated APNH (acidic proteins with affinity for nucleohistone) (18), and have studied its in vitro DNA binding properties. We have demonstrated that APNH binds to DNA optimally at physiological conditions (9-11). Equilibrium and kinetic competition experiments indicated that this protein fraction interacts preferentially with A-T rich and/or singlestranded DNA, which suggested that APNH might affect the helix-coil transition of DNA (11)

Regulation of Gene Expression in Eucaryotes

“…Such proteins would be expected to be major determinants of the location of initiation sites fo r RNA polymerase and perhaps be composed of a variety of proteins capable of destabilizing DNA (167,168). Competi tion experiments with E. coli RNA polymerase and hen oviduct RNA polymerase II indicate that these enzymes use the same region fo r initiation of RNA synthesis on chromatin.…”

Role of tryptophyl residues in the binding of gene 32 protein from phage T4 to single-stranded DNA. Photochemical modification of tryptophan by trichloroethanol

“…This photochemical reaction results in a complete quenching of the protein fluorescence. However, determination of the tryptophan content of the irradiated protein ^^oteins which bind preferentially, and in general cooperatively, to single-stranded polynucleotides and nucleic acids have been isolated from both prokaryotic and eukaryotic cells, bacteriophages (Alberts & Frey, 1970;Reuben & Gefter, 1973;Alberts et al, 1972), viruses and bacteria (Molineux et al, 1974; Van der Vliet & Levine, 1973;Yeh et al, 1976), fungus (Banks & Spanos, 1976), calf thymus (Herricks & , and rat liver (Thomas & Patel, 1976;Duguet & De Recondo, 1978). These proteins, termed single-strand binding (SSB)1 or helix-destabilizing proteins, play an essential role in the cell [see Coleman & Oakley (1980) and Héléne et al (1982) for reviews].…”

DNA-binding Proteins in Frog Virus 3-infected Cells

“…The reason these proteins were so tightly bound is not clear at present. However, proteins with similar DNA-binding characteristics have been described in vaccinia-infected cells (Soloski et aL, 1978) and for non-histone chromosomal proteins (Thomas & Patel, 1976). Eluates were also obtained from the native D N A -S e p h a r o s e column loaded with the cytoplasmic extract of cells exposed to AFV 3 alone (Table 1, Fig.…”