Abstract
Human fragile sites are weak staining gaps in chromosomes generated by specific culture conditions. The short CGG repeating DNA derived from folate-sensitive fragile sites has been shown to exclude single nucleosomes. To test whether this nucleosome exclusion model provides a general molecular mechanism for the formation of fragile sites, a different class of fragile site, the 33-base pair AT-rich repeating DNAs derived from the rare distamycin-inducible site, FRA16B, was examined for its ability to assemble single nucleosomes and nucleosome arrays using in vitro nucleosome reconstitution methods. The FRA16B DNA fragments strongly exclude nucleosome assembly only in the presence of distamycin, and increasing the number of 33-bp repeats increases the effect of distamycin in the destabilization of the nucleosome formation, suggesting a common mechanism for the formation of fragile sites.
Highlights
Fragile sites are chromosomal abnormalities in humans and are linked to the incidence of certain cancers and other severe disorders
Nucleosome-assembled FRA16B DNA Expels Nucleosomes upon Addition of Distamycin—To examine the ability of the 33-bp AT-rich FRA16B DNA repeat to assemble into nucleosomes, a 250-bp DNA fragment containing 6 copies of the FRA16B repeat was used in a nucleosome assembly reaction and analyzed by electron microscopy
The (33bpFRA16B)6 DNA, treated under the conditions described under “Experimental Procedures,” has 65% of the DNAs bound by histone octamer and a slightly better assembly ability compared with the pUC19 DNA, which shows 45% of the molecules associated with histone octamers
Summary
Plasmids and Proteins—Two complementary oligonucleotides containing 2 copies of the 33-nt AT-rich FRA16B sequence (5Ј-ATATATTATATATTATATCTAATAATATATCTAATATATTATATATTATATCTAATAATATATATA-3Ј and 5Ј-ATATTATATATATTATTAGATATAATATATAATATA TTAGATATATTATTAGATATAATATATAAT-3Ј) were synthesized, annealed to generate a duplex with 4 nucleotide cohesive ends, and ligated head-to-tail. DNA Fragments—A 267-bp fragment containing 2 copies of the 33-bp FRA16B DNA repeat was obtained by PCR amplification of a segment of the pFRA16B1 plasmid using primers from nt 5 to 22 and from nt 182 to 205 (based on the numbering in pGEM3zf(ϩ)). A 266-bp fragment containing 4 copies of the 33-bp FRA16B DNA repeat was obtained by PCR amplification of a segment of the pFRA16B2 plasmid using primers from nt 5 to 22 and from nt 116 to 139 of pGEM3zf(ϩ). In Vitro Nucleosome Reconstitution and EM—DNA fragments (3 pmol) were mixed with purified HeLa cell histone octamers (2 pmol) in a buffer containing 2 M NaCl. The salt was slowly lowered in increments of 0.1 M to a final concentration of 0.1 M by adding a solution of 20 mM Hepes, 1 mM EDTA, pH 7.5 (5 min for each step at room temperature), to form stable nucleosomes. The reverse probing steps were carried out, and the results showed no difference with the order of probing
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