Circular dichroism of histone‐bound regions in chromatin

Abstract

SynopsisNative, NaC1-treated, trypsin-treated, and polylysine-bound nucleohistones were studied in 2.5 X M EDTA, pH 8.0, using circular dichroism (CD) and thermal denaturation. Removal of histone I by 0.6 M NaCl has a much smaller effect on both A E Z Z~and Ae~,s than the removal of other histones. This indicates that histone I has less helical content and less conformational effect on the DNA in nucleohistone. By extrapolating to 10007, binding by histones other than I, the positive CD band near 275 nm is close to zero. Comparison is also made between the effects of binding by the more basic and the less basic halves of histones by trypsin-digestion and polylysinebinding experiments. Trypsin digestion of nucleohistone reduces melting band IV at 82°C much more than melting band I11 a t 72°C. However, the CD changes of Aezn and At220 induced by trypsin digestion are small, unless melting band I11 is also reduced by the use of a higher trypsin level. This implies that the less basic halves of histones, which stabilize DNA to 72°C (melting band 111), have more helical structure and are more responsible for conformational change in DNA than are the more basic halves, which stabilize DNA to 82°C (melting band IV). Polylysine binding to nucleohistone diminishes melting band I11 but has no effect on melting band IV. This binding affects only slightly the At220 of nucleohistone, indicating that polylysine interferes very little with the structure of the less basic halves of bound histones. The implications of these studies with respect to chromatin structure are discussed.

A thermal denaturation study of chromatin and nuclease-produced chromatin fragments

“…The similarity of the maximum melting temperature band of chromatin (-80°C) to that obtained with synthetic complexes (15, 5 3 , as well as its elimination by trypsin (17,56) almost certainly establishes that this transition is due to the melting of DNA bound to the N-terminal portions of the histone molecules. The melting region between 55°C and 75°C can therefore be ascribed to the melting of DNA that is only partially protein covered.…”

“…In an attempt to establish the molecular nature of the stabilization of DNA to thermally induced unfolding in chromatin many studies have been undertaken using synthetic polypeptides (51)(52)(53), protamines (53,54), individual histones (15,16,55), and altered chromatins (17,56,57). Synthetic complexes between DNA and most basic polypeptides (15, 5 1, 55), including all the individual histones except HI, exhibit a biphasic transition with one component melting near that of the constituent DNA and the other component melting at a considerably elevated temperature.…”

Changes in the circular dichroic spectrum of calf thymus solubilized chromatin caused by ultraviolet irradiation

“…The increase of 3 ~280 observed after the dose of 1 1 An analogous change in the positive CD band of DNP is induced by dissociation of proteins from DNP, especially proteins that dissociate in the range of ionic strength 0.7-1.0 M NaCI [3,4,10,14,20,22]. The increase of 3 ~280 observed after the dose of 1 1 An analogous change in the positive CD band of DNP is induced by dissociation of proteins from DNP, especially proteins that dissociate in the range of ionic strength 0.7-1.0 M NaCI [3,4,10,14,20,22].…”

“…The increase of 3 ~280 observed after the dose of 1 1 An analogous change in the positive CD band of DNP is induced by dissociation of proteins from DNP, especially proteins that dissociate in the range of ionic strength 0.7-1.0 M NaCI [3,4,10,14,20,22]. Moreover, the removal of proteins is accom-panied by a pronounced reduction of the negative band of DNP in the vicinity of 227 nm [4,10], whereas UV irradiation caused, on the contrary, its deepening (Fig. 1) would thus be equivalent to dissociation of minimally 17% of proteins [20] (related to histone H4, which exhibits the greatest change in eUipticity at 280 nm per gramme of proteins [3].…”

“…This gives evidence of a more compact arrangement of DNA in the complex on the one hand, and of an increase of the content of alpha helix in the proteins on the other [4,10,14,20,21]. This gives evidence of a more compact arrangement of DNA in the complex on the one hand, and of an increase of the content of alpha helix in the proteins on the other [4,10,14,20,21].…”