ABSTRACTnaked DNA, and that it is the cooperative binding of histone Hi that is responsible for the folding of the thin chromosome fiber. The results of four independent experimental approaches provide evidence that supports this view: (i) competition between long and short nucleosome chains for histone Hi analyzed by filter binding; (ii) the distribution of histone Hi in mixtures of long and short chromosome fibers separated by sucrose gradient velocity sedimentation; (iii) the sedimentation behavior of long chromosome fiber fragments as a function of NaCl concentration in the range of the transition; (iv) electron microscopy of chromosome fibers above and below the transition. MATERIALS AND METHODSCell nuclei were isolated from bovine lymphocytes (13) and stored in 2 mM MgCl2, 5 mM Tris.HCl (pH 7.5), and 66% (vol/vol) glycerol at -600 until needed, but not longer than 10 days. Radioactively labeled nuclei were obtained from bovine lymphocytes that had been stimulated by phytohemagglutinin P (Difco) in medium containing [3H]thymidine after 60 hr of incubation.Fragmentation of chromatin in nuclei by micrococcal nuclease (Boehringer or Worthington) was carried out at 00 in 0.2 M sucrose, 1 mM CaCl2, 5 mM Tris-HCI (pH 7.5), and either 80 or 60 mM NaCl at a concentration of 2.4-108 nuclei per ml. The digestion reaction was terminated and nuclei were lysed by gently adding the same volume of a solution containing 5 mM EDTA, 5 mM Tris-HCl (pH 7.5), and 80 or 60 mM NaCl. The nuclear debris was pelleted at 5000 X g for 8 min. The supernatant contained 50-80% of the nuclear DNA in the form of fragmented chromosome fibers. Histone-HI-depleted fiber fragments were made by the aid of tRNA (14). Histone HI was prepared by the trichloroacetic acid extraction procedure (15).The filter binding assay was carried out as described earlier (12). Stock solutions of histone H1 were diluted into 0.5 ml samples of 5 mM Tris-HCl (pH 7.5) and 40 mM NaCl containing equal weights of labeled and unlabeled histone-Hldepleted nucleosomes. After incubation for 30 min at 00, the reaction mixture was filtered through nitrocellulose membrane filters at a flow rate of 0. 1 ml-sec-'. The filters were washed three times with 0.7 ml of buffer, dried, and monitored for radioactivity. The values given are the means of three experiments; the standard deviations were less than 10% of the mean values. Filters retained 20 (+5)% of the histone-Hi-depleted nucleosome trimer (background).Sucrose gradient analyses were made by layering fragmented chromosome fiber samples (0.5 ml) on preformed linear gradients from 10 to 30% sucrose containing 1 mM sodium phosphate (pH 6.8), 0.2 mM EDTA, and NaCl at the same concentration as in the samples. Nitrocellulose tubes with 0.5 ml cushions of 86% (vol/vol) glycerol were spun in a Beckman SW 40 rotor at 3°. The gradients were analyzed with the use of a turbulence-free flow cell (ISCO).DNA sizes were analyzed electrophoretically on 1.4% agarose gels as previously described (16

A 34-mer oligonucleotide containing a single 7,8-dihydro-8-oxoguanine (8-OxoG) residue was used to study the enzymatic and DNA binding properties of the Fpg protein from E. coli. The highest rates of incision of the 8-OxoG containing strand by the Fpg protein were observed for duplexes where 8-OxoG was opposite C (*G/C) or T (*G/T). In contrast, the rates of incision of duplexes containing 8-OxoG opposite G (*G/G) and A (*G/A) were 5-fold and 200-fold slower. Gel retardation studies showed that the Fpg protein had a strong affinity for duplexes where the 8-OxoG was opposite pyrimidines and less affinity for duplexes where the 8-OxoG was opposite purines. KDapp values were 0.6 nM (*G/C), 1.0 nM (*G/T), 6.0 nM (*G/G) and 16.0 nM (*G/A). The Fpg protein also binds to unmodified (G/C) duplex and a KDapp of 90 nM was measured. The cleavage and binding of the (*G/C) duplex were also studied using bacterial crude lysates. Wild type E. coli crude extract incised the 8-OxoG containing strand and formed a specific retardation complex with the (*G/C) duplex. These two reactions were mediated by the Fpg protein, since they were not observed with a crude extract from a bacterial strain whose fpg gene was inactivated. Furthermore, we have studied the properties of 6 mutant Fpg proteins with Cys-->Gly mutations. The results showed that the 2 Fpg proteins with Cys-->Gly mutations outside the zinc finger sequence cleaved the 8-OxoG containing strand, formed complexes with the (*G/C) duplex and suppressed the mutator phenotype of the fpg-1 mutant. In contrast, the 4 Fpg proteins with Cys-->Gly mutations within the zinc finger motif neither cleave nor bind the (*G/C) duplex, nor do these proteins suppress the fpg-1 mutator phenotype.

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Chromosome fibers isolated from lymphocyte nuclei and prepared for electron microscopy by techniques designed to preserve their native structure have a distinctly knobby appearance, suggesting that DNA and protein are not distributed evenly along the fiber axis. Individual knobs (superbeads) are arranged in tandem and have an average diameter of about 200 A. Mild nuclease digestion of isolated nuclei releases apparent monomer superbeads that are composed of nucleohistone particles with the properties of nucleosomes. The kinetics of digestion indicate that the superbead is a discrete structural unit containing, on the average, about eight nucleosomes.

A DNA glycosylase that excises, 2,6-diamino-4-hydroxy-5N-methylformamidopyrimidine (Fapy) from double stranded DNA has been purified 28,570-fold from the yeast Saccharomyces cerevisiae. Gel filtration chromatography shows that yeast Fapy DNA glycosylase has a molecular weight of about 40 kDa. The Fapy DNA glycosylase is active in the presence of EDTA, but is completely inhibited by 0.2 M KCl. Yeast Fapy DNA glycosylase does not excise N7-methylguanine, N3-methyladenine or uracil. A repair enzyme for 7,8-dihydro-8-oxoguanine (8-OxoG) co-purifies with the Fapy DNA glycosylase. This repair activity causes strand cleavage at the site of 8-OxoG in DNA duplexes. The highest rate of incision of the 8-OxoG-containing strand was observed for duplexes where 8-OxoG was opposite guanine. The mode of incision at 8-OxoG was not established yet. The results however suggest that the Fapy- and 8-OxoG-repair activities are associated with a single protein.