Abstract
Centromeres are specialized chromosome domains that control chromosome segregation during mitosis, but little is known about the mechanisms underlying the maintenance of their integrity. Centromeric ultrafine anaphase bridges are physiological DNA structures thought to contain unresolved DNA catenations between the centromeres separating during anaphase. BLM and PICH helicases colocalize at these ultrafine anaphase bridges and promote their resolution. As PICH is detectable at centromeres from prometaphase onwards, we hypothesized that BLM might also be located at centromeres and that the two proteins might cooperate to resolve DNA catenations before the onset of anaphase. Using immunofluorescence analyses, we demonstrated the recruitment of BLM to centromeres from G2 phase to mitosis. With a combination of fluorescence in situ hybridization, electron microscopy, RNA interference, chromosome spreads and chromatin immunoprecipitation, we showed that both BLM-deficient and PICH-deficient prometaphase cells displayed changes in centromere structure. These cells also had a higher frequency of centromeric non disjunction in the absence of cohesin, suggesting the persistence of catenations. Both proteins were required for the correct recruitment to the centromere of active topoisomerase IIα, an enzyme specialized in the catenation/decatenation process. These observations reveal the existence of a functional relationship between BLM, PICH and topoisomerase IIα in the centromere decatenation process. They indicate that the higher frequency of centromeric ultrafine anaphase bridges in BLM-deficient cells and in cells treated with topoisomerase IIα inhibitors is probably due not only to unresolved physiological ultrafine anaphase bridges, but also to newly formed ultrafine anaphase bridges. We suggest that BLM and PICH cooperate in rendering centromeric catenates accessible to topoisomerase IIα, thereby facilitating correct centromere disjunction and preventing the formation of supernumerary centromeric ultrafine anaphase bridges.
Highlights
The centromere is a highly differentiated chromosomal structure consisting, in human cells, of a-satellite DNA repeats [1]
All the centromeres in prophase cells displayed positive staining for GFP-Bloom syndrome helicase (BLM) (Figure 1C). These results suggest that BLM is progressively loaded to the centromeres from late S phase until mitosis
We investigated whether the helicase and/or DNA binding activities of BLM were required for centromeric localization, using GM08505 BS cells stably transfected with a construct encoding either the BLM protein with an inactive helicase domain (GFP-I841T) or with both the helicase and DNA binding domains inactivated (GFP-G891E) [13]
Summary
The centromere is a highly differentiated chromosomal structure consisting, in human cells, of a-satellite DNA repeats [1]. Our interest in centromere stability began with the unexpected discovery that a helicase-like protein, PICH (Plk1-interacting checkpoint ‘‘helicase’’) and the Bloom syndrome helicase (BLM) colocalized to centromeric ultrathin DNA threads that could not be counterstained with conventional DNA dyes or antibodies against histones [4,5]. These threads were found to be common in all cultured normal cells tested, and are probably physiological structures.
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