Mapping of a human centromere onto the DNA by topoisomerase II cleavage

Abstract

We have mapped the positions of topoisomerase II binding sites at the centromere of the human Y chromosome using etoposide-mediated DNA cleavage. A single region of cleavage is seen at normal centromeres, spanning ∼50 kb within the centromeric alphoid array, but this pattern is abolished at two inactive centromeres. It therefore provides a marker for the position of the active centromere. Although the underlying centromeric DNA structure is variable, the position of the centromere measured in this way is fixed relative to the Yp edge of the array, and has retained the same position for >100 000 years.

Construction, Characterization, and Complementation of a Conditional-Lethal DNA Topoisomerase IIα Mutant Human Cell Line

“…Thus, the abnormal cell divisions seen in yeast top2 mutants (Holm et al, 1985;Uemura and Yanagida, 1986), and in vertebrate cells treated with topoII inhibitors (Downes et al, 1991;Ishida et al, 1991Ishida et al, , 1994Haraguchi et al, 2000) are consistent with the unique ability of topoII to resolve the sister chromatids catenations generated when DNA replication forks meet (Wang, 2002). TopoII has been implicated in mammalian centromere function (Floridia et al, 2000;Spence et al, 2002), and it has been suggested that topoII in yeast may play a role in centromeric chromatin structure (Bachant et al, 2002). Further work is required to understand the relationship between any such structural role, topoII-mediated chromatid decatenation, and the role of the cohesin complex (Nasmyth, 2002) in maintaining sister chromatid cohesion until anaphase (Bernard and Allshire, 2002).…”

“…What is the nature of such catenations and why do they seem to affect only a subset of chromosomes? Given the persistent association of sister centromeres after chromatid arms have separated (Losada and Hirano, 2001;Bernard and Allshire, 2002), and the known accumulation of topoII␣ protein (Rattner et al, 1996;Sumner, 1996) and topoII activity (Floridia et al, 2000;Andersen et al, 2002;Spence et al, 2002;Agostinho et al, 2004) at centromeres, one may reasonably speculate that centromeric catenations are uniquely resolved by topoII␣ and persist after anaphase onset in topoII␣-depleted cells. In the absence of cohesin-dependent centromeric cohesion, poleward forces might then be sufficient to allow near normal separation of sister kinetochores, at the expense of severe chromosomal distortions and DNA damage, resulting in the dissociation of many, but not all, sister chromosomes.…”

Human centromere repositioning “in progress”

“…In human cells, regular arrays of canonical ␣-satellite repeats are a preferred substrate for kinetochore assembly (28,29). These arrays vary in size from Ͻ200 kb to Ͼ4 Mb without evidence of any impairment of centromeric function (19); however, studies of artificially created minichromosomes and naturally occurring deleted chromosomes suggest that centromeric function depends on a relatively small subdomain of ␣-satellite (30)(31)(32). Considerable evidence also suggests that not all ␣-satellite sequences are functionally equivalent.…”

Recurrent evolution of DNA-binding motifs in the Drosophila centromeric histone

“…Centromeric histones (CenH3s) belong to the histone H3 family of proteins, based on homology in the histone fold domain, and can replace H3 in nucleosomes (4,5). However, centromeric chromatin is distinct from the rest of the genome, as demonstrated by nuclease accessibility studies in both budding and fission yeast (6)(7)(8), as well as by topoisomerase II cleavage patterns of human centromeres (9). Instead of the regular nucleosomal arrays observed in surrounding heterochromatin, centromeric DNA appears uniformly accessible to nucleases.…”