Human Fragile Site FRA16B DNA Excludes Nucleosomes in the Presence of Distamycin

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.Fragile sites are chromosomal abnormalities in humans and are linked to the incidence of certain cancers and other severe disorders. Cytologically they are defined as sites of poor staining, gaps, or DNA strand breakage in metaphase chromosomes when cells are treated under specific culture conditions. To date, more than 100 different types of fragile sites have been identified in the human genome, classified as common or rare and further divided according to the agents used to identify them in cultured cells (1). The rare fragile sites are classified into the folate-sensitive, distamycin A-inducible and bromodeoxyuridine-inducible groups, whereas the common fragile sites include the aphidicolin-, 5-azacytidine-, and bromodeoxyuridine-inducible groups (1). Although the first fragile site was discovered in 1965 (2), the molecular nature of fragile sites is not clear. It has been shown that the locations of many fragile sites are cytogenetically correlated to those of deletion and translocation breakpoints in cancer cells (3). Also, fragile sites have been mapped at the molecular level to the integration sites of oncogenic viruses (4). Therefore, it is critical to investigate the nature of the DNA sequence and structure where fragile sites are located in order to understand how viruses find the fragile site and why fragile sites are preferential loci for deletion and translocation.FRAXA, one of the folate-sensitive fragile sites, has been studied extensively because of its direct association with fragile X syndrome, one of the most common forms of inherited mental retardation (5-7). In FRAXA, as with four other folate-sensitive sites: FRA11B (8), FRA16A (9), FRAXE (10), and FRAXF (11), the DNA sequences reveal expanded CCG repeats in individuals expressing those fragile sites. To investigate the molecular basis of these fragile sites, we (12, 13) and others (14) have examined the ability of DNA containing long blocks of CCG triplet repeats to assemble into nucleosomes. Using a combination of electron microscopy (EM) 1 and competitive nucleosome reconstitution assays, we showed that expanded CCG repeats strongly exclude nucleosomes in ...

The contribution of <i>cis</i>-elements to disease-associated repeat instability: clinical and experimental evidence

“…The rare BrdU-inducible FRA10B (Hewett et al, 1998) and Distamycin-A inducible FRA16B (Yu et al, 1997) fragile sites are associated with expanded tracts of 42-and 33-bp ATrich repeats, respectively. Recent evidence from the examination of the FRA16B AT-rich repeats (Hsu and Wang, 2002) suggests that nucleosome exclusion, a suspected cis-element (see below: Section III, Nucleosomes), may also be the underlying connection between these repetitive elements and chromosomal fragile sites.…”

The rare human fragile site 16B

“…Interfamilial differences regarding the cytogenetic expression of FRA16B were consequently attributed to different repeat copy numbers. In addition, in vitro experiments with distamycin A which binds externally to AT base pairs in the minor groove of double-stranded DNA demonstrated altered rotational positioning of nucleosome assembled DNA (Brown and Fox, 1996) and nucleosome destabilization in FRA16B DNA (Hsu and Wang, 2002).…”

Rare fragile sites

“…Furthermore, in vitro studies of the 33-bp sequence amplified in FRA16B suggest that the sequence itself, particularly in the presence of distamycin A, also excludes nucleosomes and the distamycin A effect is not limited to single nucleosomes but also to nucleosome arrays (Hsu and Wang, 2002). This could explain why FRA16B can be spontaneously expressed but that distamycin A greatly enhances its expression.…”