532 zyxwvutsrqpon Biochemistry zyxwvu 1980, 19, 532-541 Dependence of Mononucleosome Deoxyribonucleic Acid Conformation on the Deoxyribonucleic Acid Length and H 1 /H5 Content. Circular Dichroism and Thermal Denaturation Studies? Mary K. Cowmant and Gerald D. Fasman* ABSTRACT: Four mononucleosome preparations were isolated from micrococcal nuclease digests of chicken erythrocyte nuclei which differed in average deoxyribonucleic acid (DNA) length and in H1 and H5 content. The circular dichroism properties of the unperturbed mononucleosome preparations and the corresponding H 1 zyxwvutsrqpo - and H5-depleted species demonstrate that the nucleoprotein spectra above 250 nm are all altered relative to protein-free DNA by the addition of a single negative band at 275 nm, similar to the band observed for @-DNA. The quantitative analysis of the *-type band intensity for any of the higher molecular weight unperturbed samples relative to core particle mononucleosomes yielded a constant number of DNA base pairs (-140) contributing to this new band. Upon removal of H1 and H5 from the mononucleosome preparations which have sufficiently long linker DNA, the @-type band intensity indicates an -30 base pair reduction in the number of core DNA base pairs contributing to the altered circular dichroism properties. The @-type band is proposed to be due The repeating subunit of chromatin, termed the nucleosome, consists of a core particle with -140 base pairs of DNA wrapped around a histone octamer and a linker DNA segment to which the very lysine-rich histone H I (and H5 in avian erythrocytes) is bound [see reviews by Kornberg (1977) and Felsenfeld (1978)l. The binding site of H1 is situated close to the core histone octamer (Bonner, 1978; Glotov et al., 1978). The linker DNA tertiary structure is sensitive to factors such as the mode of sample preparation, the solvent conditions, and the binding of H1 or H5 (Finch & Mug, 1976; Nicolini et al., 1976; Thoma & Koller, 1977). The core DNA tertiary structure is less variable. It is a superhelix of 1.75 turns, with - 80 base pairslturn with a radius of 45 A and a pitch of 28 A (Finch et al., 1977). Details of core particle DNA secondary structure (base tilt, twist, etc.) have not yet been determined, but Raman spec- troscopic studies (Goodwin & Brahms, 1978) and wide-angle X-ray scattering data (Bram, 1971) indicate that it is closely similar, if not identical, with that of B-DNA. No evidence of C-DNA structure has been found. The circular dichroism (CD1) spectrum of core particle DNA, however, is significantly altered from that of protein-free B-DNA (Ramsay-Shaw et From the Graduate Department of Biochemistry, Brandeis Univer- sity, Waltham, Massachusetts 02254. Received February 22, 1979; revised manuscript received October 31, 1979. This is Publication No. 1288 from the Graduate Department of Biochemistry, Brandeis Univ- ersity. This research was generously supported in part by Grants from the U.S. Public Health Service (GM 17533), the American Cancer Society (P-577), and the Department of Energy (EP-78-S-02- 4962.A000). *Author to whom correspondence should zyxwvutsr be addressed. G.D.F. is the Rosenfield Professor of Biochemistry. rPresent address: Eye Research, Columbia University College of Physicians and Surgeons, New York, NY 10032. to the compact DNA tertiary structure, i.e., the manner in which the DNA is wound around the histone core allowing interactions between adjacent turns of the superhelix. This interpretation implies that -30 base pairs of core DNA are removed from the unique core tertiary structure when the linker DNA is not bound by H1 or H5. The circular dichroism analysis correlates well with the thermal denaturation prop- erties of mononucleosomes. Removal of H1 and H5 causes an overall reduction in the thermal stability of both core and linker DNA. The degree of destabilization is greatest when the average DNA length is maximum. Some core DNA is lost from the highest temperature melting bands when hi- stone-free DNA is present. These results indicate two regions of different conformational and thermodynamic stability in core DNA. The length of attached linker DNA and its histone content influence the two regions of the core to differing ex- tents. al., 1974; Mandel & Fasman, 1976; Whitlock & Simpson, 1976; Olins, D. E., et al., 1977; Tatchell & Van Holde, 1977; Cowman & Fasman, 1978). Both DNA-histone and hi- stone-histone interactions are important for the maintenance of the altered DNA conformation (Olins, D. E., et al., 1977; Lilley & Tatchell, 1977; Whitlock & Simpson, 1977). It would be of great interest if the CD data could be ana- lyzed in terms of specific changes in DNA structure. An example of a situation in which this ability to interpret spectra would be important is the case of high molecular weight chromatin. The maximum CD ellipticity of DNA in chro- matin, above 260 nm, varies from a value equal to that of core particle DNA to a higher value intermediate between that of a core DNA and protein-free DNA [see, for example, Shih & Fasman (1970), Johnson et al. (1972), Spurrier & Reeck (1976), Nicolini et al. (1976), de Murcia et al. (1978), and Fulmer & Fasman (1979a)l. Chromatin with the lower el- lipticity value has been termed “native”; chromatin with the higher value has been attributed to a “disrupted” structure (de Murcia et al., 1978). These terms are not meaningful if the manner in which the DNA structure is reflected in its CD spectrum is not understood. zyxw In particular, we have recently shown that the CD spectrum of chromatin in the disrupted structure simply corresponds to the spectrum anticipated for chomatin in the “beads-on-a-string” structure commonly ob- served in electron micrographs (Cowman & Fasman, 1978). That spectrum was observed for high molecular weight chromatin isolated by a mild nuclease digestion procedure and studied in low ionic strength solvent (Fulmer & Fasman, ~~ I Abbreviations used: CD, circular dichroism; Tris, tris(hydroxy- methy1)aminomethane; Tris-EDTA, 10 mM Tris-HCI and 0.7 mM EDTA, pH 7.5. 0006-2960/80/0419-0532$01 zyxwvuts .OO/O - 0 1980 American Chemical Societv