The aim of this study was to examine the mechanism of chromosome breakage at RSZ, a MEC1-sensitive fragile site and a model for mammalian CFSs. Specifically, we tested involvement of the following possibilities, each of which had been implicated in mammalian fragile site expression: (i) errors in replication fork restart,; (ii) premature mitotic chromosome condensation; (iii) spindle tension; (iv) anaphase; or (v) cytokinesis. Evidence revealed that Top2 and condensin proteins were required for RSZ breakage; in contrast, the key proteins involved in replication fork restart, spindle tension, anaphase, or cytokinesis were dispensable.
In all eukaryotes examined to date, an essential function of Top2 and condensins is chromosome compaction 
. Although the extent of mitotic chromosome condensation in budding yeast is about two orders of magnitude less than that observed in metazoan cells (the compaction ratio is 160 in yeast versus 10,000–20,000 in metazoans; 57,58), inactivation of budding yeast Top2 or condensin subunits also results in a chromosome compaction defect 
, in agreement with the notion that the mechanism is evolutionarily conserved.
Taken together, we propose a model whereby two temporally and genetically distinguishable events mediate chromosome breakage at RSZ
s (). In WT cells, the genome duplication is complete by the end of S phase (). During mitotic prophase, the duplicated genome undergoes Top2- and condensin- dependent chromosome compaction () in preparation for its disjunction during anaphase (). In the absence of Mec1 function, replication forks stall at RSZ
s (); despite this, the cells exit S phase and proceed through the cell cycle. During prophase, the incompletely duplicated genome of mec1-4
cells becomes subjected to Top2- and condensin- dependent chromosome compaction (). We propose that internal stress generated during this process promotes the conversion of stalled forks to a DSB. The molecular mechanism underlying the catalysis of breakage is unknown, but may involve a nuclease that is yet to be identified (below). Above evidence indicates that chromosome breakage is independent of spindle tension or tension-dependent events such as anaphase or cytokinesis. This breakage is also independent of the status of sister chromatid cohesins, consistent with the report that cohesion removal, while essential for sister chromatid resolution, is dispensable for mitotic chromosome compaction 
. In the absence of Top2 or condensins (), chromosome condensation does not take place; therefore, the incompletely duplicated genome of mec1-4
cells is not subjected to the internal stress that triggers the conversion of stalled forks to DSBs. Nevertheless, the cells die, likely due to the lack of an essential Top2 or condensin function(s) 
Proposed mechanism of RSZ breakage.
With regard to the dispensability of the replication fork restart process, it is important to note that the list of candidate genes examined is not exhaustive, and therefore, we cannot rigorously eliminate its involvement based on this line of evidence. Nevertheless, our results unequivocally rule out the involvement of some of the key players in replication fork restart that had previously been implicated in breakage at different types of fragile sites (see below); the RAD52
epistasis group proteins, the Sgs1BLM
-Top3 complex, the Srs2 helicase, and the Mus81-Mms4 endonuclease 
The dispensability of spindle tension is not surprising in the light of the fact that the distribution pattern of RSZ
s is different from that of the spindle tension mediated breaks. Specifically, RSZ
s are found between active replication origins along the entire length of the chromosome except for the centromeric region (
; N. Hashash and R. Cha, unpublished data). In contrast, spindle tension-dependent DSBs tend to occur around the centromere, the locus of greatest spindle tension 
. Mammalian CFSs, like RSZ
s, are found along the chromosome arms. Furthermore, the fact that mammalian fragile sites are defined as loci of recurrent breaks or gaps on metaphase chromosome spreads, obtained from cultures treated with spindle poisons such as colchicines 
, strongly suggest that expression of mammalian fragile sites, like that of RSZ
s, occurs independently of spindle tension.
The amount of force exerted by a pair of microtubules at the centromere (i.e. 20 piconewton [pN]) is estimated to be at least an order of magnitude smaller than that required to break the chromosome (i.e. 480 pN) 
. Assuming that the intra-chromosomal stress generated during mitotic chromosome compaction is less than that generated by the spindles, it is likely that the Top2/condensin-dependent RSZ
breakage is mediated by an endonuclease. As a means to test whether Top2 was the responsible enzyme, we performed Top2 ChIP-on-CHIP analysis in MEC1
cells, reasoning that if Top2 catalyzed the cleavage, we might observe its enrichment at RSZ
s. Analysis thus far has failed to show any such enrichment, suggesting that its direct involvement was unlikely (N Hashash, R Cha, Y Katou, K Shirahege; unpublished data). Nevertheless, this observation alone does not eliminate the possibility, because Top2 may dissociate from the ends of the DSB after DNA cleavage, and therefore would not normally remain enriched at RSZ
s. Alternatively, the cleavage might be mediated by a different protein, for example, Yen1, an evolutionarily conserved Holiday junction resolvase that is activated during M phase of the cell division cycle 
or proteins involved in post replication repair 
. It is also possible that the DSBs at RSZ
s result from cleavage of single stranded DNA associated with stalled forks 
A positive role for Top2 and condensin in chromosome breakage is unexpected in light of the observations that their inactivation caused, rather than prevented, DSB formation [e.g. 
. Also surprising is the dispensability of anaphase or cytokinesis in RSZ
breakage. Upon a closer examination, however, it becomes apparent that the chromosome breakage examined in each study was at different types of fragile loci in the genome, in that they differed with respect to their structure (e.g. a hairpin or a specific protein-DNA complex), distribution (e.g. chromosome arms versus the centromeres) and/or the timing of their expression (e.g. during S phase, before anaphase, or during cytokinesis) 
. These observations provide further support for the notion that both the stability and the expression of each type of fragile sites is under a specific genetic and regulatory control 
Among the different types of fragile sites identified and characterized to date, the RSZ
appears to be the closest structural and functional homolog of mammalian fragile sites. Furthermore, among the currently proposed mechanisms of mammalian fragile site expression, the mechanism of RSZ
breakage inferred in the current study seems to be most consistent with the original definition of a mammalian fragile site, that it is a heritable locus of recurrent breaks or gaps on metaphase chromosome spreads 
. Taken together, it is tempting to speculate that the mechanism of RSZ
breakage and that of CFS expression, at least for those that are sensitive to the loss of ATR or ATM functions 
, might be conserved and that the mammalian Top2 and condensin may similarly play a role in promoting fragile site expression.