ReviewMammalian G1- and S-phase checkpoints in response to DNA damage
Introduction
It is arguable that now and then the odd genetic mutation can be a healthy event, particularly in germ cells. Such mutations complement genetic recombination in providing limited genomic plasticity necessary for the process of evolution to select favourable traits for future generations. On the other hand, less is clearly more when it comes to genetic change, and all eukaryotes have evolved a plethora of mechanisms to minimise DNA damage. The threat of excessive genetic change needs constant attention as DNA becomes damaged by inherent errors in processes such as DNA replication, as well as through genotoxic stress from reactive cellular metabolites and exogenous stimuli (e.g. ionising radiation, ultraviolet light, cigarette smoke). Our cells cope with the required monitoring and maintenance of genomic integrity by means of a complex network of DNA repair pathways 1., 2. and the so-called cell-cycle checkpoints. The latter are biochemical signalling pathways that sense various types of structural defects in DNA, or in chromosome function, and induce a multifaceted cellular response that activates DNA repair and delays cell-cycle progression 3., 4., 5., 6., 7.. When DNA damage is irreparable, checkpoints eliminate such potentially hazardous cells by permanent cell-cycle arrest or cell death.
Reflecting their distinct positions and functions within the checkpoint cascades, components of the cell-cycle checkpoints have been subclassified into DNA damage sensors, signal transducers, and effectors [4]. To ensure faithful replication and transmission of the genome and to promote survival, checkpoints fulfil at least four tasks: they rapidly induce cell-cycle delay, help activate DNA repair, maintain the cell-cycle arrest until repair is complete, and then actively re-initiate cell-cycle progression. Mechanistic elements of the first three tasks are emerging, yet the molecular basis of the recovery from checkpoint-mediated arrest remains unknown. The biological and (patho)physiological relevance of the checkpoint pathways is supported by their evolutionary conservation [4], and it is evident from the consequences of checkpoint failure. Checkpoint malfunction leads to accumulation of mutations and chromosomal aberrations, which in turn increase the probability of developmental malformations or genetic syndromes and diseases including cancer 3., 4., 5., 6., 7., 8., 9., 10., 11..
Despite the response of some checkpoint cascades to DNA damage in quiescent cells [12•], most checkpoint pathways operate only in cycling cells, which are at higher risk of fixing and propagating deleterious mutations 3., 4., 5., 6., 7., 8., 9., 10., 11.. But even among proliferating cells, the choice of checkpoint cascade(s) to be alarmed, and the outcome of such response, depends on many variables. These factors include the type, extent and duration of the DNA-damage stimulus, the type of cell cycle (meiotic versus mitotic; early embryonic versus ‘somatic’), the cell type and differentiation stage, and the position of the cell within the cell cycle. Although we are still largely ignorant of the impact of some of these variables on checkpoint control and execution, rapid advances have recently been made in understanding the molecular basis of the checkpoint pathways operating in various phases of the mitotic cycles in mammalian somatic cells. The sensors of DNA damage remain relatively obscure, and may include the Rad1–Rad9–Hus1 complex, Rad17, and possibly the large ATM and ATR kinases of the PI3K family (phosphatidyl-inositol-3-kinase), which might recognise DNA lesions through so-far elusive subunits analogous to the Ku 70/80 proteins of DNA-PK (DNA-dependent protein kinase) 4., 8., 13•.. The choice of transducers of the damage signal (the ATM/ATR and Chk1/Chk2 kinases) reflects the type of DNA damage, though some overlap between the ATM–Chk2 axis and the ATR–Chk1 axis exists 4., 5., 6., 7.. These upstream elements of the checkpoint cascades are shared by diverse cell types and cell-cycle phases. In contrast, the downstream checkpoint effectors and their final targets within the cell-cycle machinery may differ in G1, S, or G2/M phases.
In this review, we discuss the progress in elucidating the mechanisms of the mammalian DNA-damage checkpoints that guard the entry into, and progression through, the Sphase. This focus has been motivated by the recent discoveries of the molecular basis for the rapid, p53-independent initiation of the G1 checkpoint 14••., 15••., and the intra-S-phase checkpoint 16••., 17••., 18••., 19••., 20••.. Furthermore, we provide examples of potential cell-type-restricted checkpoint responses, and the evidence for cancer-promoting aberrations in the G1- and S-phase checkpoints. Finally, we highlight the conceptual significance of these new discoveries and the challenges they raise for future research.
Section snippets
G1/S control and the two-wave G1 checkpoint response
To appreciate the workings of the G1 DNA damage checkpoint(s), it is helpful to briefly consider the G1/S control. G1 phase is a period when cells make critical decisions about their fate, including the optional commitment to replicate DNA and complete the cell division cycle. Provided mitogens are available and the cellular environment is favourable for proliferation, a decision to enter S phase is made at the so-called ‘restriction point’ in mid-to-late G1 [21]. In unstressed cells, this
Rapid, p53-independent induction of the G1 checkpoint
To be effective within minutes after DNA damage, induction of the G1 block should exploit a mechanism that is poised to act, independent of transcription and protein synthesis. Recent reports suggest that pathways which fit this definition operate by targeting Cdc25A 14••., 15••., 16••.. The phosphatase activity of Cdc25A cancels the inhibitory phosphorylation of CDK2 and is essential for G1/S transition [11]. Independent of the p53 status, the abundance and activity of Cdc25A rapidly decreases
The p53 pathway and the maintenance of the G1 arrest
Under normal conditions, p53 is a highly unstable protein and its DNA binding capacity is low. After DNA damage, numerous post-translational modifications lead to stabilisation of the p53 protein and activation of its sequence-specific DNA binding 9., 30.. Only then can p53 efficiently stimulate transcription of cell-cycle inhibitors such as p21 (Fig. 2). Furthermore, the p21 protein has to accumulate to levels sufficiently high to inhibit the CDK-containing complexes, before cell-cycle
The intra-S-phase checkpoint response
In contrast to the key role of p53 in maintenance of the DNA-induced G1 arrest, no specific roles for p53 or p21 have been implicated in the control of the intra-S-phase checkpoint. This is perhaps not so surprising as the S-phase checkpoint, manifested by a decreased rate of DNA synthesis after generation of DSBs, is by definition a transient phenomenon [5]. The absence of the ‘maintenance component’ during S phase, contrary to the G1 and G2 checkpoints, might be beneficial for the cells by
G1/S checkpoint defects and cancer
Genetic instability is one of the hallmarks of cancer, and its links to aberrations in DNA repair machinery and the cell-cycle checkpoint pathways is well documented 1., 2., 3., 4., 5., 6., 7., 8., 9., 10., 11., 30., 56., 59., 60., 61.. Evidence to support this notion continues to accumulate, and here we briefly review the known, and particularly the recently identified, cancer-associated defects of the G1/S checkpoint components (Table 1).
Except for the ATR whose lack causes early embryonic
Conclusions and future directions
The crude molecular anatomy of mammalian cell-cycle checkpoints is taking shape, and we are learning rapidly about their physiology and pathology. The two-wave concept of the G1 checkpoint, the mechanistic insights into the intra-S-phase checkpoint, and the appreciation of checkpoint aberrations as important determinants of multistep tumorigenesis exemplify the recent advances in this field. One of the unifying features of the G1- and S-phase checkpoints is their joint targeting of the Cdc25A
Update
Experiments with transgenic mice by P Sicinski and colleagues [74••] greatly substantiate the concept that cyclin D1 could indeed represent an important checkpoint target in specific tissues. An excellent review on ATM- and ATR-mediated checkpoint signalling by R Abraham has recently been published [75., 76•.]. This overview also summarises the current knowledge about the sensors of damaged DNA, including the candidacy of the Rad protein family members for such function, an issue not discussed
Acknowledgements
We are grateful to JHJ Petrini, C Cordon Cardo, J Nevins, and H Nevanlinna for sharing their data before publication, and to the Danish Cancer Society, the Danish Medical Research Council, the John and Birthe Meyer Foundation, and the Nordic Cancer Union for financial support. Our apologies to colleagues whose work could only be cited indirectly in this review.
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Now in press
The work which links the ATM checkpoint kinase with E2F-regulated transcription, referred to in the text as (J Nevins, personal communication), has now been published:
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