TGGA REPEATS IMPAIR NUCLEOSOME FORMATION

Nucleosomes, the building blocks of chromatin, are responsible for DNA packaging in eukaryotic cell nuclei. They play a structural role in g enome condensation, and influence transcription and replication. Prope rties of the DNA sequence, such as curvature and flexibility, direct t he location of nucleosomes. DNA sequences that position nucleosomes ha ve been identified and rules that govern their properties have been fo rmulated. However, DNA sequences that are refractory to nucleosome for mation have been less well characterised and it is possible that they may perturb or alter chromatin structure. Here we identify such sequen ces by selecting those that refrain from nucleosome formation from a l arge pool of synthetic DNA fragments with a central region of 146 rand om base-pairs fitted with adapters for PCR amplification. These were u sed for in vitro salt-induced reconstitution of nucleosomes under ther modynamic equilibrium conditions. Fragments that did not form nucleoso mes were purified, amplified by PCR, and the reconstitution was repeat ed. After 17 rounds of negative selection, the material was highly enr iched in sequences reluctant to form nucleosomes. Cloning and sequenci ng revealed that 35% of the molecules had long repeats of TGGA, and th eir affinity for histone octamers was about half that of average DNA. (C) 1998 Academic Press.