Proc. Natl Acad. Sci. USA Vol. 79, pp. 118-122, January 1982 Cell Biology Nucleosome phasing and micrococcal nuclease cleavage of African green monkey component a DNA (nucleosomes and chromatdn/primate DNAs/DNA sequence) PHILLIP R. MUSICH*, FRED L. BROWN, AND JOSEPH J. MAIO Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461 Communicated by Harry Eagle, September 14, 1981 ABSTRACT The micrococcal nuclease cleavage of intact nu- gestion was made that these results simply reflect a DNA se- clear chromatin from African green monkey cells and ofthe com- quence specificity of the micrococcal nuclease. Nucleosome pletely deproteinized sequences was studied by using high-reso- phasing would not have to be invoked. Although phase rela- lution analytical and DNA sequencing gels and secondary restric- tionships have been observed for other repetitive and 5S DNA tion enzyme analysis. When deproteinized component a DNAwas (5, 10), some tRNA coding regions (11), gene domains (12), and used as substrate, not all phosphodiester bonds in the 172-base- regions of the simian virus 40 genome (13, 14), these results pair repeat units were cleaved with equal frequency by the nu- would also now be in doubt if micrococcal nuclease shows site clease. A distinct preference for the cleavage of A-T rather than specificity with deproteinized DNA that could give rise to spu- G-C bonds was observed: however, A+T-richness in itselfdid not rious phase relationships in chromatin. confer susceptibility to cleavage by micrococcal nuclease. The re- We performed detailed sequence analysis ofthe micrococcal sults suggested that, in deproteinized DNA, nuclease cleavage at a particular dinucleotides may be influenced more by the effect of nuclease products of completely deproteinized component adjacent sequences than by the composition of the dinucleotide. DNA. The results indicate only a limited preference of micro- In contrast to complex cleavage patterns ofthe deproteinized com- coccal nuclease for some A+T-rich regions. The presence of ponent a DNA which arose because of multiple cleavage sites in numerous sites ofcleavage ofthe naked component a sequence the repeat unit, micrococcal nuclease cleaved component a nu- by micrococcal nuclease contrasts with the single preferred site clear chromatin at one site per nucleosome repeat, near position of cleavage when the sequence is packaged in chromatin. The 126 in the nucleotide sequence. This simple chromatin cleavage results are consistent with the hypothesis that the distinctive pattern is consistent with the discrete nucleosomal structure of cleavage pattern ofcomponent a chromatin by micrococcal nu- component a in chromatin and a direct phase relationship be- clease (5, 6) reflects the specific and discrete structure of the tween the component a DNA sequence repeats and the nucleo- component a nucleosomes and a phase relationship between some protein structural repeats. the 172-bp repeat units and the nucleosomal proteins with which they are associated (5, 6). In eukaryotes, the nucleosome is the fundamental structural unit ofchromatin. The nucleolytic enzyme micrococcal nuclease MATERIALS AND METHODS has been an important tool in the characterization ofthe struc- Isolation and Labeling of Component a DNA Segments. tural properties of nucleosomes: this enzyme, together with AGM component a from strain CV-1 cells was treated in prep- pancreatic DNase I, has been used to resolve differences be- arative amounts with HindIII or EcoRI* to obtain the 172-bp tween transcriptionally active and inactive chromatin (1-3), in monomeric repeat units as described (6). The HindIII orEcoRI* chromatin undergoing repair replication (4), and between con- segments were treated with calfalkaline phosphatase and end- densed and diffuse chromatin (5, 6). A general and basic as- labeled with [y-32P]ATP (Amersham; 2300 Ci/mmol; 1 Ci = 3.7 sumption in these studies is that, other than a preference for x 10'° becquerels) and T4 polynucleotide kinase (15). The de- A+T-rich regions in the substrate (7), there is no nucleotide rivation of the secondary segments labeled at one end and the sequence specificity in the cleavage of a DNA substrate by sequence determination strategy are illustrated in Fig. 1, which micrococcal nuclease. The cleavage pattern observed when shows the relative positions ofthe submonomer segments which chromatin is digested with this nuclease thus reflects the pack- overlap and encompass the entire component a sequence (8). aging of the genomic DNA into nucleosomal structures which Micrococcal Nuclease Digests. CV-1 nuclei.were isolated in afford a relative protection to 145-200 base pairs (bp) of DNA the presence of 80 mM NaCl at 2°C as described (6). All mi- through its intimate association-with the nucleosomal proteins. crococcal nuclease (Worthington) digests of the deproteinized We observed discrete 172-bp DNA segments in micrococcal DNAs and of CV-1 nuclear chromatin were carried out at 2°C nuclease digests of African green monkey (AGM) component in 5 mM sodium phosphate, pH 7.0/80 mM NaCl/0.25 mM a chromatin (5, 6). These segments correspond to the repeat CaCl2/0. 1 mM phenylmethylsulfonyl fluoride. After micrococ- units of component a DNA which are 172 bp long (8). On the cal nuclease digestion of CV-1 nuclei, the DNA was purified basis ofsecondary restriction digests and Southern blot analysis from the digests and the chromatin a bands containing the com- ofthese segments, we interpreted these results as evidence for ponent a sequences and chromatin b bands containing bulk DNA sequence-specific phasing of component a nucleosomes DNA sequences (6) were separated from one another and iso- (5). However, this interpretation has been questioned because lated in preparative amounts by electrophoresis in 1% agarose it was observed that deproteinized component a was cleaved gels. The a band DNAs were labeled with [y-32P]ATP as de- in a nonrandom manner by micrococcal nuclease (9). The sug- scribed above for subsequent restriction analysis. In some ex- The publication costs ofthis article were defrayed in partby page charge Abbreviations: AGM, African green monkey; bp, base pair(s). payment. This article must therefore be hereby marked "advertise- * Present address: Dept. of Biochemistry, Quillen-Dishner College of ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Medicine, East Tennessee State Univ., Johnson City, TN 37614. 118 Downloaded by guest on September 28, 2021 Cell Biology: Musich et aL Proc. Natd Acad. Sci. USA 79 (1982) 119 Hindm HindM Hindm A- - I i I i EcoRI * HphI Eco RI * Hph I B * 32 - 140 ____ * 32 P-Hindm/ Eco RI * 140 2 * EcoR.I*/Hin C *- a 2P- dIm 26 i 32p-Hind m / Hph I FIG. 1. Component a restriction segments used as substrates formicrococcal nuclease cleavage and sequencing. (A) Stretch of component a DNA spanning two tandem repeats, illustrating the relative positions of the restriction sites for Hindu, EcoRI*, and Hph I. In B through D, asterisks denote the sites of the 5_-32p end-label. (a) HindJ[ monomer was cleaved with EcoRI* into two segments of 32 and 140 bp. (C) EcoPJ* monomer was cleaved byHindiLT into two segments of 140 and 32 bp. (D)Hindl monomer was cleaved withHph I into two segments of 146 and26bp. Numbers indicate the length of the segments in bp. periments, the chromatin oligomer DNA segments were iso- (Fig. 3, lanes e and f). These segments migrated as distinct lated for subsequent digestion with HindIII or EcoRI* and bands superimposed upon a background smear in nondenatur- Southern blotting with a nick-translated radioactive component ing polyacrylamide gels. This result indicated that, in contrast a DNA probe. to the results when the sequence was cleaved at about position The end-labeled component a restriction segments were 126 in chromatin, micrococcal nuclease cleaved the deprotein- treated with various amounts ofmicrococcal nuclease for 15 min ized DNA at multiple sites along the entire 172-bp repeat. at 20C. The samples were precipitated in ethanol and resus- Micrococcal nuclease treatment ofthe monomer segments gen- pended in 20 mM Tris-HCl, pH 8.4/1 mM EDTA/10% (wt/ erated by EcoRI* also yielded a complex pattern of submon- vol) sucrose for nondenaturing polyacrylamide gel analysis (6) omer segments that was distinct from that obtained with the or in 80%Yo formamide/50 mM Tris borate, pH 8.3/1 mM EDTA HindIII monomers (Fig. 3, lane g). This indicated that the pat- for analysis on DNA sequencing gels (15). Gel Analysis. Preparative and analytical electrophoresis of a b c d e f g DNA segments in nondenaturing agarose or polyacrylamide slab gels was performed as described (6). Molecular weight markers included end-labeled restriction segments obtained I - 344 from HinfI digestions of pBR322 or from double digestions (HindIll and EcoRI*) of component a DNA. The nucleotide V sequences ofthese segments, whose lengths extend from 32 to 172 1.6 kb, have been determined (8, 16). Sequence studies were -- 126 - according to standard techniques (15) in 8-20% polyacrylamide -94 gels containing 8.4 M urea. The nucleotides at which micro- - -78 coccal nuclease cleavage occurred could be mapped in the av- erage sequence by reference to parallel lanes in the gel con- _-46 taining the purine and pyrimidine cleavage products ofthe same I - DNA segment. FIG. 2. Micrococcal nuclease cleavage of component a sequences RESULTS in chromatin. Lane a: CV-1 nuclei were treated with micrococcal nu- Micrococcal Nuclease Cleavage Site of Component a Se- clease (1200 units/ml) for30 min at2C andthe purified DNA was then quences in AGM Nuclear Chromatin. Treatment of CV-1 nu- analyzed in 1% agarose gels asdescribed (6). Roman numerals indicate clei with micrococcal nuclease released two distinct populations chromatin monomer, dimer, and tetramer segments. The bands de- noted a contain component a sequences whereas the bands denoted b of nucleosomes: one contained component a segments 172 bp contain bulk DNA sequences.