Rearrangements of chromatin structure during spermatogenesis of squid

A stepwise replacement of somatic histones on sperm-specific proteins (we have termed them illexines I1 and I2) is found to occur during spermatogenesis of squid Illex argentinus [Kadura, S.N. and Khrapunov, S.N. (1988) Eur. J. Biochem. 175, 603-607]. The chromatin from nuclei of squid immature testes has a nucleosomal DNA repeat which corresponds to the nucleosomal repeat of calf thymus chromatin (195 +/- 5 bp). As spermatozoa become mature and illexine I2 accumulates in the chromatin, the nucleosomal structure of the latter disappears and chromatin compacting takes place. The chromatin DNA from squid spermatozoa is highly resistant to micrococcal nuclease action. Spectrophotometry and spectrofluorimetry were to establish that neither illexine I1 nor illexine I2 forms a globular structure in solution under any conditions studied. Illexine I2 (approx. 7 kDa) shows a high affinity to DNA and remains bound to it under conditions when complexes of illexine I1 (approx. 9 kDa) and salmine (approx. 4.5 kDa) with DNA completely dissociate. This fact, allowing for a similar content (about 75%) of arginine in illexine I2 and salmine, suggests high clustering of arginine residues in the composition of illexine I2. It is suggested that the initial stage of histone substitution with illexine I1, which has a more moderate affinity to DNA than illexine I2, prepares chromatin for the formation of a highly packed structure by illexine I2 during squid spermatogenesis.     

Circular dichroism studies of the interaction of a limited hydrolysate of T4 gene 32 protein with T4 DNA and poly[d(A-T)].poly[d(A-T)].

gp32 I is a protein with a molecular weight of 27 000. It is obtained by limited hydrolysis of T4 gene 32 coded protein, which is one of the DNA melting proteins. gp32 I itself appears to be also a melting protein. It denatures poly[d(A-T)].poly[d(A-T)] and T4 DNA at temperatures far (50-60 degrees C) below their regular melting temperatures. Under similar conditions gp32 I will denature poly[d(A-T).poly[d(A-T)] at temperatures approximately 12 degrees C lower than those measured for the intact gp32 denaturation. For T4 DNA gp32 shows no melting behavior while gp32 I shows considerable denaturation (i.e., hyperchromicity) even at 1 degree C. In this paper the denaturation of poly[d(A-T)].poly[d(A-T)] and T4 DNA by gp32 I is studied by means of circular dichroism. It appears that gp32 I forms a complex with poly[d(A-T)]. The conformation of the polynucleotide in the complex is equal to that of one strand of the double-stranded polymer in 6 M LiCl. In the gp32 I DNA complex formed upon denaturation of T4 DNA, the single-stranded DNA molecule has the same conformation as one strand of the double-strand T4 DNA molecule in the C-DNA conformation.     

Studies on interaction between histone V (f2c) and deoxyribonucleic acids.

Histone V (2fc) from chick erythroctes was used in the study of its interaction with DNA from various sources. Complexes between this histone and DNA were formed using the procedure of continuous NaCl gradient dialysis in urea. Two physical methods, namely thermal denaturation and circular dichroism (CD), were used as analytical tools. Thermal denaturation of nucleohistone V with chick or calf thymus DNA shows three melting bands: band I at 45-50 degrees corresponds to free base pairs; band II at 75-79 degrees, and band III at 90-93 degrees correspond to histone-bound base pairs. In histone-bound regions, there are 1.5 amino acid residues/nucleotide in nucleohistone V. In contrast, a value between 2.9 and 3.3 was determined for nucleohistone I (fl) (H. J. Li (1973), Biopolymers 12, 287). Similar melting properties have been observed for histone V complexed with bacterial DNA from Micrococcus luteus. Histone V binding to DNA induces a slight transition from a B-type CD spectrum to a C-type spectrum. Trypsin treatment of nucleohistone V reduces melting band III much more effectively than band II. Such a treatment also restores DNA to B conformation in the free state. Reduction of the melting bands of nucleohistone V by polylysine binding follows the order of I greater than II greater than III, accompanied by the increase of a new band at 100 degrees. When two bacterial DNAs of varied A + T (adenine + thymine) content simultaneously compete for the binding of histone V, the more (A " T)-rich DNA is selectively favored. Under experimental conditions described here, Clostridium perfringens DNA with 69% A + T is bound by histone V in preference to chicken DNA with 56% A + T although the latter has natural sequences for histone V binding.     

AMPHIPHILIC β-SHEET PEPTIDES CAN BIND TO DOUBLE AND TRIPLE STRANDED DNA

It was shown that synthetic peptides with amphiphilic β-sheet structure can bind to and stabilize double and triple stranded DNA. CD spectra indicated that β-sheet conformation of peptides were emphasized in the presence or absence of DNA and that no significant change of DNA conformation occurred. UV melting study at pH 7.0 revealed that interaction of peptides with DNA and its hybrids are sensitive and specific depending the host structure.     

Amphiphilic beta-sheet peptides can bind to double and triple stranded DNA

It was shown that synthetic peptides with amphiphilic beta-sheet structure can bind to and stabilize double and triple stranded DNA. CD spectra indicated that beta-sheet conformation of peptides were emphasized in the presence or absence of DNA and that no significant change of DNA conformation occurred. UV melting study at pH 7.0 revealed that interaction of peptides with DNA and its hybrids are sensitive and specific depending the host structure.     

The structure of nucleosome core particles as revealed by difference Raman spectroscopy.

Raman spectra have been observed of nucleosome core particles (I) prepared from chicken erythrocyte chromatin, its isolated 146 bp DNA (II), and its isolated histone octamer (H2A+H2B+H3+H4)2 (III). By examining the difference Raman spectra, (I)-(II), (I)-(III), and (I)-(II)-(III), several pieces of information have been obtained on the conformation of the DNA moiety, the conformation of the histone moiety, and the DNA-histone interaction in the nucleosome core particles. In the nucleosome core particles, about 15 bp (A.T rich) portions of the whole 146 bp DNA are considered to take an A-form conformation. These are considered to correspond to its bent portions which appear at intervals of 10 bp.     

Probing structural factors stabilizing antisense oligonucleotide duplexes: NMR studies of a DNA.DNA duplex containing a formacetal linkage.

The duplex formed by annealing the formacetal backbone modified dodecamer d-(CGCGTTOCH2OTTGCGC) to its complementary strand, d(GCGCAAAACGCG) (duplex I), has been studied by NMR techniques and analyzed with reference to its unmodified counterpart (duplex II). Comparison of parameters such as 2D cross-peak intensities, coupling constants, and spectral patterns indicates that structural perturbations caused by the incorporation of the formacetal linkage are minimal and localized to the central T4.A4 block. Duplex I adopts a B-type helical conformation with regular Watson-Crick base pairing and normal minor groove width. The methylene group is accommodated along the phosphate backbone in a conformation similar to that of the PO2 group found in the B-form DNA family. The central T6-T7 base pairs of duplex I melt simultaneously with the duplex, indicating a cooperative transition to single strands. Although the formacetal linkage affects global melting, as evidenced by a 3 degree C reduction in Tm for duplex I with respect to duplex II, the present study indicates that this is not the result of localized premelting at the formacetal site of duplex I but rather reflects the subtle interplay of several structural and energy factors which need to be further explored.     

Nucleotide sequence binding preferences of nogalamycin investigated by DNase I footprinting

Four DNA restriction fragments, designated tyrT, pTyr2, pUC13, and Xbs1, have been used as substrates for footprinting studies with DNase I in the presence of the anthracycline antibiotic nogalamycin. With each fragment a distinct pattern of antibiotic-protected binding sites is observed, but no concensus sequence emerges from the data. All sites are located in regions of alternating purine-pyrimidine sequence, most commonly associated with the dinucleotide steps TpG (CpA) and GpT (ApC), suggesting that the preferred binding sites may contain all four nucleotides and/or that peculiarities of the dynamics of DNA conformation at alternating sequences may be critical for nogalamycin binding. Some concentration dependence of footprinting patterns is evident, in contrast to previous studies with a variety of sequence-specific ligands. Enhanced susceptibility to attack by DNase I is commonly observed at sequences flanking strong antibiotic-binding sites. Nogalamycin selectively inhibits cleavage of DNA at certain guanine-containing sequences by the G-specific photosensitized reaction with methylene blue. Comparison of these effects with its action on the G-specific reaction with dimethyl sulfate suggests that the amino sugar moiety of nogalamycin may be preferentially located in the minor helical groove at some binding sites but in the major groove at others.     

Interactions between N- and C-terminal domains of the Saccharomyces cerevisiae high-mobility group protein HMO1 are required for DNA bending

The Saccharomyces cerevisiae high-mobility group protein HMO1 is composed of two DNA-binding domains termed box A and box B, of which only box B is predicted to adopt a HMG fold, and a lysine-rich C-terminal extension. To assess the interaction between individual domains and their contribution to DNA binding, several HMO1 variants were analyzed. Using circular dichroism spectroscopy, thermal stability was measured. While the melting temperatures of HMO1-boxA and HMO1-boxB are 57.2 and 47.2 degrees C, respectively, HMO1-boxBC, containing box B and the entire C-terminal tail, melts at 46.1 degrees C, suggesting little interaction between box B and the tail. In contrast, full-length HMO1 exhibits a single melting transition at 47.9 degrees C, indicating that interaction between box A and either box B or the tail destabilizes this domain. As HMO1-boxAB, lacking only the lysine-rich C-terminal segment, exhibits two melting transitions at 46.0 and 63.3 degrees C, we conclude that the destabilization of the box A domain seen in full-length HMO1 is due primarily to its interaction with the lysine-rich tail. Determination of DNA substrate specificity using electrophoretic mobility shift assays shows unexpectedly that the lysine-rich tail does not increase DNA binding affinity but instead is required for DNA bending by full-length HMO1; HMO1-boxBC, lacking the box A domain, also fails to bend DNA. In contrast, both HMO1 and HMO1-boxAB, but not the individual HMG domains, exhibit preferred binding to constrained DNA minicircles. Taken together, our data suggest that interactions between box A and the C-terminal tail induce a conformation that is required for DNA bending.     

Problems and paradigms: The active role of DNA as a chromatin organizer

Histone octamers (hos) and DNA topoisomerase I contribute, along with other proteins, to the higher order structure of chromatin. Here we report on the similar topological requirements of these two protein model systems for their interaction with DNA. Both histone octamers and topoisomerase I positively and consistently respond to DNA supercoiling and curvature, and to the spatial accessibility of the preferential interaction sites. These findings (1) point to the relevance of the topology-related DNA conformation in protein interactions and define the particular role of the helically phased rotational information; and (2) help to solve the apparent paradoxical behaviour of ubiquitous and abundant proteins that interact with defined DNA sites in spite of the lack of clear sequence consensuses. Considering firstly, that the interactions with DNA of both DNA topoisomerase I and histone octamers are topology-sensitive and that upon their interaction the DNA conformation is modified; and secondly, that similar behaviours have also been reported for DNA topoisomerase II and histone H1, a topology-based functional correlation among all these determinants of the higher order structure of chromatin is here suggested.     

DNA-binding studies of XSPTSPSZ,derivatives of the intercalating heptad repeat of RNA polymerase II

The synthesis, solution conformation, and interaction with DNA of three 8-residue peptides structurally related to the heptad repeat unit found at the C-terminus of RNA polymerase II are reported. Peptides QQ, XQ, and PQ are derived from the parent sequence YSPTSPSY (peptide YY), which was reported to bind to DNA by bisintercalation [M. Suzuki (1990) Nature, Vol. 344, pp. 562–565], and contain either a 2-quinolyl (Q), 2-quinoxolyl (X), or 5-phenanthrolyl (P) group in place of the aromatic side chains of the N- and C-terminal tyrosine residues present in the parent sequence. The combined results of linear dichroism and induced CD measurements of peptides QQ, XQ, and PQ with calf thymus DNA are consistent with weak binding of the peptides to DNA in a preferred orientation in which the chromophores are intercalated. Small increases in the melting temperatures of poly[d(A-T)₂] are also consistent with the peptides interacting with DNA. While enzymatic footprinting with DNase I showed no protection from cleavage by the enzyme, chemical footprinting with fotemustine showed that the peptides modify the reactivity of the major groove, presumably via minor groove binding. Peptide QQ inhibited fotemustine alkylation significantly more than either XQ or PQ, and slightly more than YY. In aqueous solution, nmr experiments on QQ, XQ, and PQ show a significant population of a conformation in which Ser2-Pro3-Thr4-Ser5 form both type I and type II β-turn conformations in equilibrium with open chain conformations. Nuclear magnetic resonance titration experiments of PQ with (GCGTACGC)₂ showed small changes in chemical shifts, consistent with the formation of a weak nonspecific complex. Analogous experiments, using peptides QQ and XQ with (GCGTACGC)₂, and peptide YY with (CGTACG)₂, showed no evidence for the interaction of the peptides with these oligonucleotides. These results show that peptides of general structure XSPTSPSZ are weak nonspecific DNA binders that differ significantly from previously characterized S(T)PXX DNA-binding motifs that are generally AT-selective minor groove binders. © 1997 John Wiley & Sons, Inc. Biopoly 42: 387–398, 1997     

Comparison between histones FV and F2a2 of chicken erythrocyte. II. Interaction with homologous DNA.

The conformation and stability of artificial complexes between chicken erythrocyte DNA and homologous histones FV and F2a2 was studied by circular dichroism (CD) and thermal denaturation followed by both absorbance and CD measurements. The complexes are made after a stepwise potassium fluoride gradient dialysis without urea and studied at low ionic strength (10-minus 3 M). 1) No structural changes of the DNA can be detected up to r equals 0.2 with FV and r equals 0.6 for F2a2. With FV at higher values of r the CD spectrum is altered, indicating the organization of DNA and histones in some kind of aggregate. 2) The conformation of histone molecules inside the complexes is not related to the ionic strength of the medium but to an effective ionic environment close to 0.1 M. This ionic strength would also correspond to the melting temperature of histone-covered DNA. 3) From the analysis of the absorbance melting profile the length of DNA covered with an histone molecule can be estimated. A good agreement is found between the negative charge of this piece of DNA and the net positive charge of the histone. 4) Since the CD transition at 227 nm occurs before the second absorbance transition at 280 nm, the DNA is stabilized no longer by native histone but partially or fully denatured histones. The helical regions of the histone molecule are not involved in the binding process, which appears to be almost purely coulombian and most likely related to some structural fit between the pattern of negative charges in the DNA helix and that of positive charges along the peptide chain.     

Studies on the structure of chemically methylated DNA

CD and melting temperature measurements on the nature of DNA with chemically methylated guanine-rich sites indicate that the stable secondary structure of DNA depicted by Ramstein et al- involves considerable distortions resulting from decreased base-base stacking interaction. Besides that quantum chemical data gained from PPP calculations are in favor of a weaker hydrogen bonding interaction in the methylated guanine-cytosine base pair. CD measurements demonstrate that methylated DNA-regions differ from the nonmethylated helical structure, since formation of a condensed conformation as occurs in the transition from B to the C-uke structure is prevented by positively charged methylated guanine residues. An increase in helix winding angle, however, can not be excluded. Binding ability of the dyes acridine orange, phenosafranine, and the antibiotic actinomycin C is lowered for methylated DNA, while binding of proflavine is, in accordance with the results of Ramstein and Leng, slightly enhanced. The reason for the opposite behavior of proflavine is at present not fully understood. In particular changes in the binding ability with dyes could not be correlated with base specificity of complex formation. It is discussed that structural changes in DNA towards a loose conformation decrease the binding tendency for acridine orange, phenosafranine, and actinomycin C.     

Noncovalent interaction of G-quadruplex DNA with acridine at low concentration monitored by MALDI-TOF mass spectrometry

The telomeric G-rich single-stranded DNA d(T(2)G(8)) can adopt in vitro G-quadruplex structure, even at low DNA concentration. Studies on stability of telomeric structures, has gained importance recently as the molecules, which can stabilize quadruplex structure, can inhibit cancer progression. In this study, G-quadruplex structure is formed by 1.0 mM NH(4)(I) ion. Stability of G-quadruplex complex is studied on interaction with acridine using CD and MALDI-TOF mass spectrometry. MALDI-TOF mass spectrometric experiments were carried out mainly to observe the noncovalent drug-DNA interactions at low concentration. From MALDI-TOF spectrum, it is identified that three ammonium ions are required for the formation of G-quadruplex structure and to provide stability to NH(4)(I)-G-quadruplex complex. With MALDI-TOF it is evident that two acridine molecules interact with NH(4)(I) G-quadruplex complex. CD studies, shows that stability of NH(4)(I) G-quadruplex, decreases and conformation change takes place on interaction with acridine. Interaction with drug reduces mostly due to transformation of G-quadruplex complex to single stranded DNA.     

The influence of the 2-amino group of guanine on DNA conformation. Uranyl and DNase I probing of inosine/diaminopurine substituted DNA.

The conformation of the DNA helix is supposed to be a critical element in site-specific recognition by ligands both large and small. Groove width is one important measure of the conformation which varies with the local nucleotide composition, perhaps because of the presence of a purine 2-amino group on G.C base pairs. We have probed DNA with G-->inosine (I) and/or A-->diaminopurine (DAP) substitutions to see whether the location of the purine 2-amino group can indeed affect the minor groove width. At acid pH, the reactivity towards uranyl nitrate is modulated in substituted DNA quite differently from natural DNA, consistent with a marked narrowing of the minor groove at sites of G-->I substitution and widening at sites of A-->DAP replacement. The latter exerts the dominant effect. The expected changes in conformation are equally evident in the patterns of susceptibility to DNase I cleavage, but not to hydroxyl radical attack. Nuclease cleavage is maximal in normal and substituted DNA at regions of inferred moderate groove width which are generally little affected by the nucleotide substitutions. Consistent with models of sequence-dependent cutting by DNase I we find that the presence of a purine 2-amino group on the base pair three places upstream of the cutting site has a profound influence on the rate of reaction.     

The effect of low ionic strength on the radiation chemistry and physical properties of calf thymus DNA

Studies of ultraviolet and circular dichroism spectra of aqueous solutions of calf thymus (CT) DNA confirm the tendency of DNA to change conformation at low ionic strength. The qualitative shape and transition width of 260 nm melting curves below 1 mM NaCl differed significantly from those previously published for DNA solutions containing 1 mM NaCl and above. Neutral aqueous solutions of CT DNA at low ionic strengths (0.1 mM-10 mM NaCl) were irradiated with low doses of gamma-rays. The melting temperature, Tm, of irradiated DNA samples increased below 1 mM NaCl suggesting interstrand crosslinking of the denatured DNA or formation of regions of more thermally stable DNA conformation. The magnitudes of these radiation responses were found to be a function of the time elapsed between salt concentration changes and irradiation as well as time after irradiation. These results are consistent with the hypothesis that the purine and pyrimidine base chromophores in double stranded DNA are sheltered from radical attack by the sugar phosphate backbone. Low dose radiation studies (0.8-8.0 Gy) of CT DNA in 1 mM NaCl and below showed a split dose and dose rate dependence for the sample melting curves.