Sedimentation velocity studies have been carried out with isolated double-strand DNA fragments prepared by digestion of PM2 phage with the restriction endonuclease Hae III. The results show that DNA molecules shorter than about 200 base pairs behave almost exactly as rigid rods with a diameter of 27 A. The behavior of the larger fragments (up to 1735 base pairs) can be described very well by either the theory of Yamakawa and Fujii (Yamakawa, H., and Fujii, M. (1973), Macromolecules 6. 407) using the same diameter and a persistence length of 575 A, or the theory of Hearst and Stockmayer (Hearst, J.E., and Stockmayer, W.H. (1962), J. Chem. Phys. 37, 1425) using a diameter of 20 A and a persistence length of 525 A.

The DNA in intranuclear yeast chromatin is protected from rapid staphylococcal nuclease degradation so as to yield an oligomeric series of DNA sizes. The course of production and disappearance of the various oligomers agrees quantitatively with a theory of random cleavage by the enzyme at uniformly susceptible sites. The sizes of the oligomers are integral repeats of a basic size, about 160 base pairs, and 80-90% of the yeast genome is involved in this repeating structure. Within this repeat there exists a 140 base pair core of more nuclease-resistant DNA. During the course of digestion, the sizes of the oligomers decrease continuously. The widths of the distribution of DNA sizes increase in order: monomer (1 X repeat size, half width = 5-7 base pairs) less than dimer (2 X repeat size, half width = 30 base pairs) less than trimer (3 X repeat size, half width = 40-45 base pairs). The yeast genome thus seems to have variable spacing of the nucleaseresistant cores, to produce the average repeat size of about 160 base pairs. Also, the presence of more than one species of monomer and dimer at certain times of digestion suggests a possible heterogeneity in the subunit structure.

Micrococcal nuclease digestion of intact chicken erythrocyte nuclei is shown to result in the formation of core nucleoprotein particles containing about 140 base pairs of DNA. These core particles, which are almost entirely devoid of histones fI and f2c, are derived from transient nucleoprotein particles containing an average of approximately 180 base pairs of DNA. Oligomers of these latter particles may be isolated after brief nuclease digestion. (2). The latter has been proposed by Kornberg (9) to be the basic DNA repeating unit associated with an eight-histone complex. We will show that the eight-histone complex is associated with 140 base pairs of DNA, and that the remaining DNA in the subunit is especially nuclease-sensitive. MATERIALS AND METHODSIsolation and Digestion of Erythrocyte Nuclei. Blood was obtained from adult White Leghorn chickens by cardiac puncture in the presence of heparin. After centrifugation at 3000 X g for 10 min, the plasma and buffy coat were removed. The erythrocytes were washed twrice with an isotonic saline solution and frozen at -600 until needed. The frozen erythrocytes were thawed at 370 in an equal volume of 0.15 M NaCl, 0.015 M Na citrate, pH 7.2 (saline/citrate) and centrifuged at 3000 X g for 10 min; the nuclear pellet was resuspended in 0.25% Nonidet P-40 in saline/citrate. In some experiments 1 mM phenylmethane sulfonyl fluoride was added to inhibit protease action (15). The nuclei were repelleted, washed with saline/citrate and resuspended in 0.3 M sucrose, 0.75 mM CaCl2, 10 mM Tris-HCl, pH 7.2 at a concentration of 2 X 108 nuclei per ml. Digestion of the nuclei by micrococcal nuclease (Worthington) was carried out at 370 with 125 units of nuclease per ml of nuclei suspension. The digestion reaction was terminated by making the solution 10 mM in EDTA, 0.15 M NaCl, and 1% sodium dodecyl sulfate (NaDodSO4). After pronase treatment (0.5 mg/ml) for 4 hr at 370 the DNA was extracted by an NaDodSO4-phenol procedure (16).When the digested chromatin was to be fractionated on an agarose A-Sm column, the reaction was terminated by the addition of EDTA to 10 mM and cooling on ice. After centrifugation at 12,000 X g for 15 min, the nuclei were resuspended in 10 ml of 10 mM Tris-HCI, pH 7.5, 0.7 mM EDTA and disrupted by homogenization for about 1 min at a medium setting on a Virtis homogenizer. The nuclear debris was pelleted at 10,000 X g for 15 min. The supernatant was made 7% in sucrose and applied to a Bio-Rad A-Sm column, 90 X 2.5 cm, equilibrated with 10 mM 0

We have compared the chromatin subunit structure of yeast, HeLa, and chicken erythrocyte by analyzing the DNA fragments produced by in situ digestion with staphylococcal nuclease (EC 3.1.4.7) and DNase I (EC 3.1.4.5). The repeat size of the chromatin varies among (and within two of) the three organisms but the size and the structure of the most nuclease-resistant "core" of the repeat is the same. Thus, the interspecies differences in repeat size are due to different lengths of nuclease-sensitive "spacer" DNA between the cores. There also seems to be a difference in the manner of spacing of cores; the transcriptionally active (yeast and HeLa) chromatins have spacings of variable length while the transcriptionally inactive (chicken erythrocyte) has a more regular spacing of cores. Intranuclear digestion of eukaryotic chromatins by staphylococcal nuclease produces a series of discretely sized DNA fragments (oligomers) with sizes that are integral multiples of a basic size (monomer) (1). Evidence indicates that this pattern of fragments results from partial protection of the DNA by a repetitive organization of histones (2,3), which appear in the electron microscope (4, 5) as globular particles [termed nucleosomes (6)], spaced along the chromatin strands.The size of the DNA contained in monomeric and oligomeric nucleoprotein fragments decreases with digestion time; monomers are digested to relatively stable "core particles" containing about 140 base pairs (bp) of DNA and eight histone molecules (7,8). It was suggested for chicken erythrocyte (8) that this behavior reflects the existence of two well defined DNA domains within the repeating unit: a relatively nuclease resistant "core" DNA fragment bound tightly to the eight histones, and a nuclease sensitive "spacer" domain which is distinguished by its relative accessibility to nuclease digestion.To decide whether observed interspecies repeat size differences (1, 7, 9-11) are real differences and whether the core and spacer distinction occurs in genetically active organisms, we have carried out detailed comparative studies of the staphylococcal nuclease digestion of chromatin from three quite different cell types: chicken erythrocytes, HeLa cells, and baker's yeast. These three encompass the range of higher and lower eukaryotes and transcriptionally inactive (erythrocyte) and active (yeast and HeLa) chromatins. We find that the size and structure of the core is similar in all three organisms but the length of the nuclease sensitive DNA between the cores and the mode of spacing varies. MATERIALS AND METHODSPreparation of Nuclei and Nuclease Digestion. Nuclei were isolated and resuspended in digestion buffer as previously described for yeast (10), chicken erythrocyte (8), and HeLa (12) with the following exceptions: for all organisms, 0.1 mM phenylmethylsulfonyl fluoride was added to cell lysis buffer and again during resuspension in digestion buffer; for HeLa cells, lysis buffer contained 1 mM CaCl2, digestion buffer contained 0.1 mM CaC12, and the nuclei were...

A chimeric metallopeptide derived from the sequences of two structurally superimposable motifs was designed as an artificial nuclease. Both DNA recognition and nuclease activity have been incorporated into a small peptide sequence. P3W, a 33-mer peptide comprising helices alpha2 and alpha3 from the engrailed homeodomain and the consensus EF-hand Ca-binding loop binds one equivalent of lanthanides or calcium and folds upon metal binding. The conditional formation constants (in the presence of 50 mM Tris) of P3W for Eu(III) (K(a) = (2.1 +/- 0.1) x 10(5) M(-1)) and Ce(IV) (K(a) = (2.6 +/- 0.1) x 10(5) M(-1)) are typical of isolated EF-hand peptides. Circular dichroism studies show that 1:1 CeP3W is 26% alpha-helical and EuP3W is up to 40% alpha-helical in the presence of excess metal. The predicted helicity of the folded peptide based on helix length and end effects is about 50%, showing the metallopeptides are significantly folded. EuP3W has considerably more secondary structure than our previously reported chimeras (Welch, J. T.; Sirish, M.; Lindstrom, K. M.; Franklin, S. J. Inorg. Chem. 2001, 40, 1982-1984). Eu(III)P3W and Ce(IV)P3W nick supercoiled DNA at pH 6.9, although EuP3W is more active at pH 8. CeP3W cleaves linearized, duplex DNA as well as supercoiled plasmid. The cleavage of a 5'-(32)P-labeled 121-mer DNA fragment was followed by polyacrylamide gel electrophoresis. The cleavage products are 3'-OPO(3) termini exclusively, suggesting a regioselective or multistep mechanism. In contrast, uncomplexed Ce(IV) and Eu(III) ions produce both 3'-OPO(3) and 3'-OH, and no evidence of 4'-oxidative cleavage termini with either metal. The complementary 3'-(32)P-labeled oligonucleotide experiment also showed both 5'-OPO(3) and 5'-OH termini were produced by the free ions, whereas CeP3W produces only 5'-OPO(3) termini. In addition to apparent regioselectivity, the metallopeptides cut DNA with modest sequence discrimination, which suggests that the HTH motif binds DNA as a folded domain and thus cleaves selected sequences. The de novo artificial nuclease LnP3W represents the first small, underivatized peptide that is both active as a nuclease and sequence selective.