J. Mol. Biol. (1995) 245, 559–567 Poly(dA)·Poly(dT) Forms Very Stable Nucleosomes at Higher Temperatures Henry L. Puhl and Michael J. Behe The synthetic polymer poly(dA)·poly(dT) was long thought to be refractory Department of Chemistry Lehigh University, Bethlehem to nucleosome formation. Several years ago our laboratory demonstrated that PA 18015, U.S.A. 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°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. Keywords: nucleosome; poly(dA)·poly(dT); histone; reconstitution Introduction Most DNA in eukaryotic cells is packaged into the nucleoprotein complexes called nucleosomes. This fact shows that nucleosome formation is compatible with a large number of DNA sequences. However, it has long been recognized that if there existed in the nucleus a subset of sequences with structural properties different from bulk DNA, then preferential binding of nucleosomes to, or exclusion from, such sequences could occur. Such altered interactions could then cause nucleosomes surrounding the sequence to be phased with respect to the sequence. In turn, phasing of nucleosomes could contribute to the regulation of gene expression by causing a promoter to be covered or uncovered, by sterically interfering with the binding of regulatory proteins, or by other, more indirect, methods. A number of candidates for the role of ‘‘phasing element’’ have been investigated over the past decade, including Z-DNA (Garner & Felsenfeld, 1987), cruciforms (Nickol & Martin, 1983), and flexible DNA (Shrader & Crothers, 1989, 1990). Another category of potential phasing elements is that composed of oligopurine·oligopyrimidine tracts. It has been known for several decades that synthetic double-stranded polydeoxynucleotides in which one strand is composed completely of purines and the other strand is composed completely of pyrimidines have structures that are different from heterogeneous-sequence DNA. X-ray fiber diffrac- tion studies (Leslie et al ., 1980) have shown that, although heterogeneous-sequence DNA from almost any source and synthetic DNA with an alternating purine-pyrimidine sequence have nearly identical diffraction patterns, that of the classical B-form, poly(dA)·poly(dT), poly(dI)·poly(dC), and poly[d(AI)]·poly[d(CT)] all have somewhat different helical parameters. The circular dichroism spectra of poly(dA)·poly(dT), poly(dG)·poly(dC) and poly[d(AG)]·poly[d(TC)] are quite distinctive and different from the expected circular dichroism spectra for polymers in a normal B conformation (Wells et al ., 1970). The plausibility of a role for (Pu·Py) n sequences in the modification of chromatin structure was increased by two observations. First, the number of long (Pu·Py) n (ne10) tracts in eukaryotic, but not prokaryotic, DNA is significantly greater than expected on statistical considerations alone, and they are much more abundant than sequences that could potentially form Z-DNA or other unusual DNA structures (Behe, 1987). Second, both poly(dA)·poly(dT) and poly(dG)·poly(dC) were originally reported to resist nucleosome formation (Rhodes, 1979; Simpson & Ku ¨ nzler, 1979). Thus it seemed likely that DNA containing shorter tracts of (Pu·Py) n could at least decrease the free energy of nucleosome formation. 0022–2836/95/050559–09 $08.00/0 7 1995 Academic Press Limited