Although ATM and ATR may collaborate in many ways, the organismal and cellular phenotypes that result from the loss of ATM and ATR are dramatically distinct: the loss of ATR causes rapid lethality at the earliest embryonic stages2, 3
, whereas people with no functional ATM can live for decades. What then is the essential function of ATR? As already detailed, ATR responds primarily to primed ssDNA implying that the formation of this structure in unperturbed cell cycles is at the heart of the matter. One can thus envision at least two answers to this question.
One possibility is that ATR is activated at low levels in every cell cycle as a result of normal Okazaki fragment-dependent lagging strand synthesis, during which the activating structure may be repeatedly produced. This activation may be required to enforce the order of S and M phase and to control origin timing5
. In yeast, however, separation of function mutants indicate that Mec1/ATR’s essential function has little to do with cell cycle arrest or origin firing, but is related to its fork stabilization activity143
The second possibility is that there are abundant sources of stalled forks in unperturbed cells due to endogenous damage and sequences that are difficult to replicate. ATR would thus be needed to stabilize these structures and promote their restart when collapse occurs. In effect then, the essential function of ATR may be to respond to DNA damage or replication stress, just as it does when cells are stressed by a DNA damaging agent or replication inhibitor. On this point, it should be noted that the essential function of Mec1/ATR can be rescued in unstressed cells by mutations that increase deoxynucleotide levels144, 145
. Since Mec1/ATR regulates deoxynucleotide production by regulating ribonucleotide reductase, the loss of Mec1/ATR may create the very conditions (stalled forks) that require Mec1/ATR to be resolved.
Although these are not mutually exclusive models, it seems likely that the second possibility is true. After all, ATR did not evolve to respond to the doses of radiation and chemicals used in the laboratory to study signaling. DNA is a fairly stable molecule, but the large amount of it in the nucleus (3 billion base pairs for human cells) provides ample opportunities for chemical modifications such as nicks, base modifications and crosslinks146
. Repair enzymes constantly correct these lesions but undoubtedly some are encountered by replication forks prior to repair. In addition, some DNA, DNA-RNA, and DNA-protein structures may stall replication even in the absence of any change in nucleic acid chemistry147–153
. In some of these cases, the helicase may not uncouple from the polymerase so ATR would not be activated, but in many circumstances, uncoupling does occur and the checkpoint is required for fork stabilization. Even on the relatively small circular E. coli
genome it is estimated that nearly all replication forks encounter lesions and must be repaired during every round of replication even in normal growth conditions154
. For mammals, sister chromatid exchange rates suggest that approximately 10 DSBs per cell division form at replication forks155
. The frequency of fork stalling is undoubtedly much higher.
Defects in ATR-dependent activities at stalled forks may be a major cause of genetic instability. Stalled forks are usually not problematic in eukaryotic cells with multiple replication origins since a fork converging from the other direction can complete replication. In the absence of ATR, however, a stalled fork will collapse and double-strand breaks accumulate156
. Chromosomal fragile sites may be examples of this phenomenon. Elevated rates of fragile site breakage are observed when cells are treated with low doses of replication stress agents. Therefore, fragile sites are thought to represent chromosomal regions that are particularly difficult to replicate or perhaps are difficult to repair if replication forks collapse in these areas157
. ATR-deficient cells have high levels of fragile site breakage158
. Similar fragile sites are thought to exist in S. cerevisiae
cells and are associated with slow replicating zones that have high rates of fork stalling especially in Mec1/ATR-deficient cells159
. Thus, the consequences of defective ATR-dependent replication regulation are DSBs and, eventually, cell death.