Tracts of adenosine and cytidine residues in the genomes of prokaryotes and eukaryotes.

Large segments of the S. cerevisiae, C. elegans, D. melanogaster, mouse, and human genomes, as well as the genomes of four bacterial species, have been analyzed for the occurrence of tracts of separated, alternating, and mixed adenosine and cytidine residues. Several surprising features have been observed. Although both yeast and nematode DNA are rich in AT base pairs, the genomes of these organisms have widely different biases for long homonucleotide tracts. Yeast has many long tracts of oligoadenosine, while C. elegans has an extraordinary abundance of oligocytidine tracts. Tracts of alternating A-C residues are overrepresented in most eukaryotic organisms examined. Tracts of mixed adenosine and cytidine residues, however, are especially frequent in the human genome.

Oligoadenosine tracts favor nucleosome formation.

We have measured the ability of oligoadenosine tracts 25 base pairs in length to influence nucleosome formation. Such tracts can cause DNA to bind in nucleosomes at higher temperatures with a free energy up to 1 kcal/mol more favorable than heterogenous-sequence DNA. Furthermore, the position of the oligoadenosine tract affects the free energy of binding, with the most favorable position occurring at the dyad axis.

Non-conservative mutations are well tolerated in the globular region of yeast histone H4.

Yeast histone H4 has been mutagenized at several positions which participate in the globular core of the nucleosome. The native protein contains residues at those positions which are invariant or highly conserved over all known H4 sequences, whether from yeast, Tetrahymena or higher eukaryotes. Nonetheless the protein is tolerant of non-conservative mutations. At the level of cell function the mutant proteins cause no significant change in length of the cell cycle of mating efficiency. At the level of chromatin structure no effect is observed on the internucleosomal spacing of chromatin or the pattern of hydroxyl radical cleavage of nucleosomal DNA.

An overabundance of long oligopurine tracts occurs in the genome of simple and complex eukaryotes.

A search of sequence information in the GenBank files shows that tracts of 15-30 contiguous purines are greatly overrepresented in all eukaryotic species examined, ranging from yeast to human. Such an overabundance does not occur in prokaryotic sequences. The large increase in the number of oligopurine tracts cannot be explained as a simple consequence of base composition, nearest-neighbor frequencies, or the occurrence of an overabundance of oligoadenosine tracts. Oligopurine sequences have previously been shown to be versatile structural elements in DNA, capable of occuring in several alternate conformations. Thus the bias toward long oligopurine tracts in eukaryotic DNA may reflect the usefulness of these structurally versatile sequences in cell function.

Poly(dA).poly(dT) forms very stable nucleosomes at higher temperatures.

The synthetic polymer poly(dA).poly(dT) was long thought to be refractory to nucleosome formation. Several years ago our laboratory demonstrated that the polymer could be mixed with authentic nucleosomes in a low-salt exchange procedure to form a nucleoprotein complex that behaved in a manner identical with that of nucleosomes. Competitive exchange assays at 37 degrees C showed that the homopolymer reconstituted about as well as heterogenous-sequence DNA. However, studies by other laboratories have shown that the conformation of poly(dA).poly(dT) depends on temperature; the polymer converts from its well-known, atypical structure, found at ambient temperature, to a conformation more closely resembling a canonical B form as temperature is increased. We have measured the ability of the homopurine.homopyrimidine to form nucleosomes as a function of temperature. It is seen that poly(dA).poly(dT) forms nucleosomes more strongly as the temperature of the exchange mixture is increased, so that poly(dA).(dT) outcompetes heterogeneous-sequence DNA for histones at elevated temperatures.

Structure of nucleosomal DNA at high salt concentration as probed by hydroxyl radical.

The structure of a 146 base-pair nucleosomal DNA has been probed using hydroxyl radical cleavage in buffers containing NaCl concentrations ranging from 80 mM to 800 mM. The highest salt concentrations used here are close to those required to dissociate core histone H2A and H2B from nucleosomal DNA. Nonetheless, the cleavage pattern of the DNA is unchanged over the tenfold salt concentration range, retaining the approximately 10.0 base-pairs per turn helical periodicity in the flanking regions and approximately 10.7 base-pairs per turn periodicity in the central dyad region that is characteristic of nucleosomal DNA. The rotational frame of the DNA is similarly unaffected by salt. These results support the contention that the differential free energy of bending of DNA around the nucleosome is independent of salt concentration.

Isolated oligopurine tracts do not significantly affect the binding of DNA to nucleosomes.

Nucleosomal-length DNA was constructed to contain one of two 10 bp oligopurine-oligopyrimidine sequences, either d(A10.T10) or d(G10.C10). The 146 base pair (bp) sequences were then each tandemly cloned. This allowed for the production of circularly-permuted sequence variants in which the oligopurine tract was located at eight different positions. The permuted sequences were then assayed for their ability to reconstitute into nucleosomes by competitive reconstitution. The results of the assay indicate that the free energy of nucleosome formation differs only by several tenths of a kilocalorie per mole for an oligopurine tract at any position along the DNA, including the central dyad region.

Poly[d(A.T)] and other synthetic polydeoxynucleotides containing oligoadenosine tracts form nucleosomes easily.

Synthetic double-stranded polydeoxynucleotides of the general form poly[d(AnT).d(ATn)], with n ranging from 3 to 11, have been synthesized. The conformation of the polymers was investigated by circular dichroism spectroscopy and the polymers were examined for their ability to form nucleosomes. Although spectra show that a circular dichroism band characteristic of poly[d(A.T)] appears in the polymer family for n greater than 7, we demonstrate that even polynucleotides with the longest tracts of contiguous adenosine bases (n = 11) are able to form nucleosomes when reconstituted using a histone exchange procedure. Thus resistance to nucleosome formation does not coincide with the appearance of features similar to that of poly[d(A.T)] over the bulk of the nucleosomal DNA. Furthermore, we show that an approximately 150 base-pair poly[d(A.T)] itself, long thought to be refractory to nucleosome formation, can assemble into such a protein-DNA complex when reconstituted by a low-salt exchange procedure. Competitive assays show that the homopolymer reconstitutes about as well as heterogeneous sequences DNA. Our work, therefore, suggests that highly adenosine-rich sequences in vivo apparently have a function that operates at a level other than that of nucleosome structure.

Eukaryotic DNA does not form nucleosomes as readily as some prokaryotic DNA.

Nucleosomal-length DNA was prepared from the genomic DNA of various prokaryotic and eukaryotic organisms by limited nuclease digestion after reconstitution with core histones. The DNAs ranged in base composition from 26.5% to 72% guanosine-plus-cytosine (%GC). The nucleosomal-length DNAs were then used in a competitive reconstitution assay in order to quantitatively determine their relative abilities to form nucleosomes. The results of the assay indicate a linear dependence of the free energy of nucleosome formation on base composition and, surprisingly, show that several prokaryotic DNAs form nucleosomes as well as or better than eukaryotic DNAs.

The protein-folding problem: the native fold determines packing, but does packing determine the native fold?

A globular protein adopts its native three-dimensional structure spontaneously under physiological conditions. This structure is specified by a stereochemical code embedded within the amino acid sequence of that protein. Elucidation of this code is a major, unsolved challenge, known as the protein-folding problem. A critical aspect of the code is thought to involve molecular packing. Globular proteins have high packing densities, a consequence of the fact that residue side chains within the molecular interior fit together with an exquisite complementarity, like pieces of a three-dimensional jigsaw puzzle [Richards, F. M. (1977) Annu. Rev. Biophys. Bioeng. 6, 151]. Such packing interactions are widely viewed as the principal determinant of the native structure. To test this view, we analyzed proteins of known structure for the presence of preferred interactions, reasoning that if side-chain complementarity is an important source of structural specificity, then sets of residues that interact favorably should be apparent. Our analysis leads to the surprising conclusion that high packing densities--so characteristic of globular proteins--are readily attainable among clusters of the naturally occurring hydrophobic amino acid residues. It is anticipated that this realization will simplify approaches to the protein-folding problem.

Oligopurine.oligopyrimidine tracts do not have the same conformation as analogous polypurine.polypyrimidines.

The ability of tracts of synthetic oligopurine.oligopyrimidines containing both adenosine and guanosine residues to approach the conformation of analogous polypurine.polypyrimidines has been examined as a function of tract length by CD spectroscopy. Tracts of up to 19 contiguous, alternating dA and dG residues yield CD spectra that are distinctly different from that of the analogous alternating polymer. Thus the structural changes reflected in the unusual CD spectrum of poly[d(AG)].poly[d(CT)] must require even longer tract lengths. Tracts of contiguous adenosines flanked by guanosine residues were seen to approach the CD spectrum of poly[dA].poly[dT] quite slowly as a function of tract length, requiring more than 24 contiguous adenosines to give CD spectra similar to the homopolymer. These results lead us to the conclusion that oligopurine tracts in vivo are not well modeled by synthetic polypurine.polypyrimidines with one or two base pair repeating units.

Co-polymer tracts in eukaryotic, prokaryotic, and organellar DNA.

Large variations in DNA base composition and noticeable strand asymmetries are known to occur between different organisms and within different regions of the genomes of single organisms. Apparently such composition and sequence biases occur to fulfill structural rather than informational requirements. Here we report the wide occurrence of a more subtle biasing of DNA sequence that can have structural consequences: an increase or a suppression of the number of long tracts of two-base co-polymers. Strong biases were observed when the DNA sequences of the longest eukaryotic, prokaryotic, and organellar entries in the GenBank data base (totaling 773 kilobases) were analyzed for the number of occurrences of tracts of the two-base co-polymers (A,T)n, (G,C)n, and (A,C)n as a function of tract length. (The expression (A,T)n is used here to denote an uninterrupted tract, n nucleotides in length, of A and T bases in any proportion or order, terminated at each end by a G or C residue.) Characteristic differences are also observed in tract biases of eukaryotic vs. prokaryotic organisms.

Histone deletion mutants challenge the molecular clock hypothesis.

A basic tenet of the molecular clock hypothesis is that the rate of sequence drift for a protein depends on the number of amino acid residues that are critical for its function. However, recent experiments have determined that, although core histone sequences are highly conserved among eukaryotes, large regions of the proteins are dispensable for growth in yeast.

The B-Z transition in supercoiled DNA depends on sequence beyond nearest-neighbors.

In order to examine sequence-dependent structural effects in DNA, the ability of alternating purine-pyrimidine fragments to undergo a B-Z transition when cloned in a supercoiled plasmid was determined solely as a function of sequence, with base and nearest-neighbor composition held constant. Sequences of 22 GC and 2 AT base pairs were synthesized such that the AT base pairs varied between contiguous placement and separation by eight GC base pairs. Results show, surprisingly, that the ease of the B-Z transition varies with the position of the two AT base pairs, occurring at lower superhelical densities when AT base pairs are contiguous, and at higher torsional strain when the AT base pairs are moved further apart.

Competitive nucleosome reconstitution of polydeoxynucleotides containing oligoguanosine tracts.

The ability of synthetic polydeoxynucleotides composed of oligoguanosine tracts of increasing length to form nucleosomes has been determined by several reconstitution procedures. When the presence of nucleosomes is determined by resistance to nuclease digestion, a protected band of approximately 150 base-pairs is detected only with difficulty for polymers containing long tracts of contiguous guanosines. However, when assayed by a shift in the electrophoretic mobility of radiolabeled polymers exchanged at 0.7 M-NaCl with authentic nucleosomes, all polymers tested are seen to form nucleosomes. Quantitative competitive reconstitution shows that the length of the tracts per se does not adversely affect their propensity to form nucleosomes, since even 150 base-pair poly(dG).poly(dC) forms nucleosomes as well as heterogeneous-sequence DNA. However, the ability to form nucleosomes does depend on the length of the polymer repeating unit.

Nucleosome reconstitution of core-length poly(dG).poly(dC) and poly(rG-dC).poly(rG-dC).

The double-stranded polypurine.polypyrimidines poly(dG).poly(dC) and poly[d(A-G)].poly[d(T-C)] and the mixed ribose-deoxyribose polynucleotide poly(rG-dC).poly(rG-dC) have been successfully reconstituted into nucleosomes. The radioactively labeled particles comigrate in gel electrophoresis and sucrose density gradient experiments with authentic nucleosomes derived from chicken erythrocyte chromatin. These results show that nucleosomes are able to accommodate a wider variety of polynucleotides than was previously believed.