Bloom’s Syndrome and PICH Helicases Cooperate with Topoisomerase IIα in Centromere Disjunction before Anaphase

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.

The Smc5/6 Complex Is an ATP-Dependent Intermolecular DNA Linker

“…Taken together with the herepresented in vitro analysis, this could suggest that Smc5/6 facilitates Top2-dependent formation of SCIs. However, reduction of the function of either Smc5/ 6 or Top2 leads to the formation of unresolved DNA bridges at anaphase (Gallego-Paez et al, 2014;Rouzeau et al, 2012;Wang et al, 2008), indicating a function for Smc5/6 in the resolution of SCIs. Accordingly, Smc5/6 accumulates on S. cerevisiae chromosomes after inhibition of Top2 (Jeppsson et al, 2014;Kegel et al, 2011).…”

SUMO-interacting motifs (SIMs) in Polo-like kinase 1-interacting checkpoint helicase (PICH) ensure proper chromosome segregation during mitosis

“…This difference 34 and inhibition of BLM has been previously shown to cause chromatin bridges. 26,35 Additionally, PICH is a SUMO substrate itself 10 and it may be possible that SUMOylation can cause conformational changes through intra-molecular interaction between SUMOylated PICH and SIM1&2 (Fig. 5).…”

PICH promotes mitotic chromosome segregation: Identification of a novel role in rDNA disjunction

“…Using RNAi-based protein depletion approaches, a consensus has emerged that PICH is required for chromosome maintenance, but some phenotypic variation has been reported following PICH depletion. 8,[15][16][17][18][19][20] In an effort to clarify any discrepancies, our laboratory, in collaboration with the Nigg and Wang laboratories, generated chicken and human cell lines completely lacking PICH via targeted gene inactivation. 21,22 Consistent with a role for PICH in faithful genome maintenance, the PICH-deficient chicken cells had increased levels of chromatin bridging, micronuclei, and polyploidy.…”