DNA is not unlike the finest bone china — subject it to stress and it will break. One particular stress is caused by conditions that partially inhibit DNA replication (folate deficiency, for example, or treatment with aphidicolin), and in this case the breaks occur preferentially at specific loci called common fragile sites. These sites — which form breaks and gaps on metaphase chromosomes — are rearranged in many tumours, hence the interest in knowing how their stability is regulated.

Reporting in Cell, Thomas Glover, graduate student Anne Casper and colleagues show that the replication checkpoint kinase ATR (ataxia-telangiectasia and Rad3-related) is a crucial factor in fragile-site stability. The authors were led to look at this kinase because ATR — along with the related ATM (ataxia-telangiectasia, mutated) — is implicated in the response to DNA damage during cellular replication.

To test the idea that ATM and ATR might be involved in fragile-site stability, Glover and co-workers first looked at the effects of 2-aminopurine (2-AP), which inhibits the kinase activities of both proteins. Although fragile-site gaps and breaks were seen when human lymphocytes were treated with aphidicolin alone, the addition of 2-AP led to a dramatic rise, with over 90% of these gaps occurring at known fragile sites.

Glover and colleagues next asked whether both ATM and ATR are involved. To do this they first compared fragile-site stability in control cell lines versus AT lymphoblast cell lines with truncating mutations in ATM. There was little difference in the stability of fragile sites in the normal and mutant cell lines, even after treatment with aphidicolin, suggesting that ATM is not involved in fragile-site stability.

ATR, by contrast, is crucial, as shown by several experiments. One difficulty is that ATR animals are not viable, so the authors first used a dominant-negative approach. They tested the effects of inducing fragile sites with aphidicolin in human osteosarcoma cells stably transfected either with wild-type ATR or with a kinase-dead form (ATR-kd). They found that, after the addition of aphidicolin, the number of chromosome breaks and gaps increased more than 20-fold in the cells expressing ATR-kd compared with those expressing wild-type ATR. Glover and colleagues confirmed this finding using two other approaches — cre–lox-mediated inactivation of ATR, and RNA interference (RNAi) against ATR. In both cases, the appearance of gaps and breaks increased in response to aphidicolin in the ATR-deficient cells.

These results indicate that, during DNA replication, stalling of the replication fork at fragile sites could be involved in the instability of these sites, and the known replication-checkpoint function of ATR is consistent with a role for this protein. So, could stalling at fragile sites be a normal occurrence during cellular replication? The authors reasoned that, if this were the case, they might observe fragile-site instability in ATR-deficient cells in culture, without the need for aphidicolin. When they tested this over five days, they found up to a 30-fold increase in chromosome breaks and gaps in the ATR-deficient cells compared with wild type.

The authors propose, then, that fragile sites represent unreplicated single-stranded chromosomal regions, which result from stalled replication forks that escape the ATR-dependent replication checkpoint. They also predict that tumours with alterations in the replication checkpoint, or in the homologous-recombination machinery that compensates for such problems, might show increased chromosome rearrangements at fragile sites.