Fragile sites are especially sensitive to drugs that interfere with S phase. The group found that these same sites are also susceptible to a drug called calyculin A, which induces immediate chromatin condensation. DNA breaks appeared primarily when cells were treated with the drug during S phase or in G2. The further into G2 the drug was added, the fewer breaks were found, suggesting that whatever marks the sites as fragile was progressively corrected before mitosis.
The authors suspect that this “mark” is unreplicated DNA (or replicated DNA that has not yet regained its histone complement). Fragile sites mapped to the transitions between early- and late-replicating sequences, which might might act as barriers to fork progression. As such, fragile sites may be the last sequences replicated (even doing so in G2 rather than S phase) and thus be especially sensitive to premature condensation.
If they are the last to be copied, fragile sites would make ideal spots for G2/M checkpoint proteins to monitor. ATR, for instance, which stabilizes stalled forks, might prevent condensation until fragile sites are replicated. Indeed, fragile sites in cells lacking ATR have been shown to break more often than in normal cells. The authors are now using calyculin A to determine whether ATR sits on unreplicated fragile sites.