While the canonical mechanism driving chromosome segregation is via kinetochore-microtubule interactions, studies have demonstrated efficient segregation of chromosomes lacking centromeric DNA (Ishii et al., 2008
; Kanda et al., 2001
; Kaye et al., 2004
; Platero et al., 1999
). This occurs either through the formation of neocentromere or direct association of the acentric to intact chromosomes. Our analysis of I-CreI-induced acentrics reveals a distinct tether-based mechanism by which acentrics are efficiently segregated to daughter cells. These acentrics rely on DNA threads decorated with BubR1, Polo, INCENP, and Aurora-B to segregate equally toward the poles ().
Model for the Role of Tether in Acentric Segregation
The observation that segregating acentrics possess a DNA tether connecting them to their centric partner suggests that tethers facilitate acentric segregation. During the period in which segregation of the acentric is delayed while the main mass of chromosomes has fully segregated, the length of the tether increases to accommodate the increased distance between the segregating centric fragment and the inert acentric. This increase could occur either through a spooling-out to create a longer tether, or stretching of the tether. We favor the latter alternative, as this readily explains the delay in acentric segregation followed by prompt segregation to the poles until they reach the main mass of chromatids. That is, the tether may be elastic, and as tension builds, the tether stretches ultimately resulting in rapid tether contraction (). Elastic forces have been proposed in other instances in which chromatin tethers have been observed. Severing of crane-fly meiotic chromosomes during anaphase results in the acentric chromosome fragments moving backward across the equator (LaFountain et al., 2002
). This finding led to the conclusion that sister telomeres are connected by an elastic tether that exerts a force opposing poleward forces. In Drosophila
, heterochromatic threads connect achiasmatic chromosome homologs during meiosis (Hughes et al., 2009
). It is proposed that these threads mediate congression of nonexchange chromosomes via their elastic properties.
Reduction of BubR1 or Polo activity results in an increase in the frequency of abnormal acentric segregation and a decrease in acentric chromosome tethering. These observations indicate that these tether-associated kinases are involved in tether function. They may play a role in generating tether-elastic forces driving acentric segregation. This idea is supported by the observation that, during anaphase, in bubR1- and polo-compromised mutants, acentrics linger at the metaphase plate much longer than acentrics in wild-type cells. We also find instances in these mutants where tethers stretch from the inert acentrics to the segregating chromosome mass without initiating acentric poleward movement. This suggests a failure in tether contraction.
Although a large number of cells in larval brains exhibit lagging acentric chromosomes after I-CreI induction, there is no effect on adult survival. Insight into the high survival rates comes from the finding that in a wild-type background, sister acentrics segregate accurately to opposing poles with a high frequency. Thus, if a cell enters anaphase with a DSB, a final option may be to properly segregate acentrics enabling reassociation of the centric and acentric chromosome fragments and repair of the DSB in the daughter cells. This is supported by the observation that in bubR1
mutants, the frequency in which acentrics segregate equally to opposing poles is significantly decreased and there is a corresponding reduction in adult survival. We cannot rule out that spindle defects inherent to the polo
mutants underly the synthetic lethality of polo
mutants with I-CreI expression. However, we found that mutations disrupting BubR1 kinase activity, which alters spindle structure, produce a less dramatic defect in acentric segregation than mutations in the BubR1 KEN box that impair BubR1 checkpoint function. The fact that spindle integrity was not detectably altered in a previous analysis of bubR1-KEN
mutant neuroblasts (Rahmani et al., 2009
) indicates that abnormal spindle structure is not the primary cause of acentric segregation defects in BubR1-compromised mutants and its corresponding synthetic lethality with I-CreI expression. Moreover, this suggests that BubR1 spindle checkpoint function plays a role in efficient acentric segregation. In undamaged cells, BubR1 localizes at kinetochores and inhibits the anaphase-promoting complex/cyclosome (APC/C) until all chromosomes are properly attached to the spindle (Musacchio and Salmon, 2007
). Recently, BubR1 has been found to accumulate on unprotected telomeres and is thought to activate the spindle checkpoint (Musarò et al., 2008
). We found that I-CreI-generated acentrics delay anaphase onset via activation of the DNA damage checkpoint Grp(Chk1) but independently of the BubR1 spindle checkpoint activity (Royou et al., 2005
). We speculate that some as-yet unidentified APC/C substrates are associated with the tether and are important for tether function. Since BubR1 remains strongly associated with the tether well into anaphase, it may efficiently inhibit the APC/C activity locally on the tether, thus preserving tether integrity throughout mitosis. On the other hand, BubR1 KEN box may have a role in addition to APC/C inhibition that is important for BubR1 function on the tether.
We currently do not know the complete nature of the DNA tethers reported here and the mechanisms by which they form. The fact that tether can form in euchromatin as well as heterochromatin indicates they are a general feature of Drosophila
chromosomes. We speculate that the presence of DBSs may result in the cell entering mitosis with unresolved replication intermediates that promote tether formation. Support for this idea comes from recent studies reporting that replication stress results in the formation of BLM (Bloom syndrome, RecQ helicase-like)-associated ultrafine DNA bridges linking homologs at fragile loci during mitosis (Chan et al., 2009
). An alternative possibility is that the presence of DSBs even after replication has terminated necessitates the long-term recruitment of the repair machinery. The cell may enter mitosis with repair intermediates hampering chromatin condensation at the site of DSBs, thus creating the tethers. Interestingly, DNA tethers form between chromosome homologs or heterologs during meiosis when condensin complex function is impaired (Hartl et al., 2008
). The observation that BubR1 accumulates on DNA breaks regardless of the chromatin state suggests a more direct role of BubR1 on DNA repair in mitotic cells. It might, for instance, stabilize the repair machinery that keeps the DNA fragments apposed.
We find that tethered and untethered acentric sister chromatids remain tightly apposed well into anaphase. The mechanism by which these sisters are held together is unclear as cohesins are removed from chromosome arms as early as prophase in Drosophila
(Warren et al., 2000
). Similar observations have been shown in yeast in which centric and acentric fragments were created by the HO endonuclease (Kaye et al., 2004
; Melo et al., 2001
). The authors found instances where acentric sister chromatids remain linked. This association depends partially on repair machinery components and impairs their proper segregation. Based on these findings, we speculate that I-CreI-generated acentric sister chromatids are held together by the entanglement of their respective tethers generated by repair mechanisms. In most instances, this entanglement is resolved during progression through anaphase. Failure to resolve entanglement would result in the unequal segregation of acentrics ().
Recent studies have reported DNA tether-like structures that connect achiasmate chromosomes in Drosophila
meiosis (Hughes et al., 2009
). These threads contain the passenger proteins INCENP and Aurora B. INCENP-coated DNA tethers are also present during anaphase in mammalian cells (Baumann et al., 2007
; Wang et al., 2008
). Significantly Aurora B and INCENP decorate I-CreI-induced tethers. This implies that all tethers share similar properties and their origin and structure are conserved features of the eukaryotic cell cycle.