Doi:10.1016/j.mrfmmm.2003.09.004

DNA replication, the cell cycle and genome stability The 16 reviews and original research articles in of this special issue, Sidorova and Breeden view this special issue of Mutation Research provide an the G1 to S transition and point out that successful overall view of our current understanding of the re- completion of this transition is essential for genome lationships between DNA replication, the cell cycle stability. They suggest that successful completion of and genome stability in eukaryotic cells. As you will S-phase requires the establishment of a dual S-phase discover as you read these articles, problems with surveillance system, which, by measuring both the DNA replication—when origins fire inefficiently, or number of pre-RCs and the number of replication when precursors are in short supply, or when poly- forks, monitors ongoing DNA replication. According merases do not work properly, or when other enzymes to their hypothesis, when the number of pre-RCs and of DNA replication are faulty, or when DNA is dam- replication forks is below a threshold level, check- aged, or when checkpoint pathways are defective, or point activation will not be sufficiently robust to even (but less frequently) when all systems are work- signal in trans that late origins should not fire or ing optimally—are a major source of mutations and that mitosis should be restrained until completion of genomic rearrangements. Indeed, it seems likely that S-phase. Thus, if cells attempt to enter S-phase before most of the genomic instability that is necessary for the an adequate number of pre-RCs has been generated in development of many types of cancer can ultimately G1, the number of pre-RCs and forks may fall below be attributed to replication problems.
the threshold, leading to genomic instability. For this All of the laboratories represented in this special reason, it seems plausible that the acceleration of the issue previously made important contributions, pub- G1 to S transition evident in most cancer cells may lished elsewhere, to our understanding of replication be directly responsible for some or all of the genomic and genome stability. Their additional contributions, published here in a single issue, further advance that Exactly how the number of pre-RCs and the num- understanding and provide a convenient single source ber of replication forks is monitored by checkpoint for readers who wish to be apprised of the current state surveillance mechanisms is not yet clear. However, considerable evidence, both from yeasts (reviewed Preparations for DNA replication begin in late M-phase and continue into G1-phase, when the ori- cells (reviewed by Kim et al. indicates that the gin recognition complex (ORC), Cdc6, Cdt1 and DDKs, which are essential for initiation of replication other proteins cooperate to load the minichromosome at origins, also play upstream and downstream roles maintenance (MCM) proteins onto chromatin to form in S-phase checkpoints. Within the DDK regulatory pre-replication complexes (pre-RCs) at sites that have subunit (Dbf4), distinct motifs have been identified the potential to become replication origins. At the be- that are more important for checkpoint function than ginning of S-phase, cyclin-dependent kinase (CDK) for replication function, and it is possible that DDK and Cdc7 kinase (also called Dbf4-dependent kinase function may be required to restart stalled replica- (DDK)) cooperate to signal initiation of DNA repli- tion forks as well as to initiate fork movement in the cation at a subset of the pre-RCs. In the first article first place However, not all checkpoint functions 0027-5107/$ – see front matter 2003 Elsevier B.V. All rights reserved.
doi:10.1016/j.mrfmmm.2003.09.004 Editorial / Mutation Research xxx (2003) xxx–xxx are dependent on DDKs. In murine embryonic stem replication forks arrested at cyclobutane pyrimidine cells, complete loss of Cdc7 protein leads to ap- dimers that is inhibited by caffeine.
parent checkpoint responses—rapid cessation of Some of the major pathways by which cells re- DNA synthesis and cell proliferation, followed by store stalled forks employ homologous recombination.
Helleday gantly summarizes how homologous Many of the reviews and research articles in this recombination can be used to restore both collapsed special issue deal with the ways in which cells re- replication forks, where one of the parental strands has spond to replication fork blockage by DNA damage, been broken to create a “1-end” double-strand break, or to replication fork stalling due to weakened DNA and stalled forks, that may have re-wound to generate polymerases, or to inadequate supply of precursors branched “chicken-foot” structures.
(deoxynucleoside triphosphates (dNTPs)). Check- Major contributions to replication fork preser- point responses appear to provide the first line of vation and re-starting are also made by the set of defense. Longhese et al. provide an extensive pathways that, for historic reasons, has been called overview of these checkpoints, with a focus on check- “post-replication repair”. Now we know that these points in Saccharomyces cerevisiae. Kai and Wang pathways employ several mechanisms for bypassing view S-phase checkpoints in the fission yeast, DNA lesions that would otherwise stall replication Schizosaccharomyces pombe, and McGowan forks. Since the lesions are bypassed and not repaired, centrates on S-phase checkpoints in mammalian cells.
perhaps a better name for these pathways would be Since there is extensive conservation of S-phase “DNA damage bypass”. Smirnova and Klein checkpoints among all eukaryotic organisms, these three views are both complementary and mutually re- tween the “error-free” bypass pathways, which in most cases employ recombination mechanisms, and The checkpoint kinases ATR and ATM (in mam- the “error-prone” pathways, which in most cases in- malian cells), Mec1 (in budding yeast) and Rad3 (in volve a small amount of non-faithful DNA synthesis fission yeast) play essential roles in sensing problems by one or more translesion polymerases. Smirnova with DNA molecules and in transducing signals to and Klein point out that even in the absence downstream checkpoint pathways. These kinases can of external sources of DNA damage, the error-free be effectively inhibited in vitro by caffeine in the mil- bypass pathways cooperate with checkpoint and ho- limolar concentration range. Kaufmann et al. mologous recombination pathways in diploid yeast port that, consistent with previous observations, 2 mM cells to prevent genomic instability. Barbour and Xiao caffeine reversed ATM- and ATR-dependent S and G2 checkpoints in immortalized human fibroblasts.
recombination pathways are defective, an alterna- Mysteriously, however, 2 and even 5 mM caffeine tive damage avoidance pathway, which involves the were unable to reverse ATM-dependent checkpoint protein Mgs1, permits lesion bypass in some cases.
function in G1-phase One possible interpretation Replication forks can stall when DNA polymerases of this observation is that caffeine may not be able to have difficulties, even in the absence of DNA damage.
inhibit ATM or ATR inside living cells, even though it can inhibit these kinases in vitro, and the observed which polymerase difficulties are induced by growing inhibition of S and G2 checkpoints by caffeine may fission yeast cells that harbor a temperature-sensitive indicate that caffeine is able to inhibit other steps polymerase ␣ at an elevated, but not lethal, tempera- (not ATM or ATR) in these checkpoint pathways.
ture. Under these conditions, the mutation frequency While caffeine was able to inhibit the S checkpoint is increased. The higher mutation frequency appears response to UVC, its effect on UVC genotoxicity was to be due, at least in part, to a small amount of DNA rather small in normal human fibroblasts. In contrast, synthesis by an error-prone translesion polymerase in xeroderma pigmentosum variant cells, which lack that is loaded onto replication forks by the checkpoint the damage-bypass polymerase DNA pol ␩, caffeine clamp loader (Rad17) and the checkpoint clamp com- markedly enhanced UVC genotoxicity, suggesting plex of Rad9, Rad1 and Hus1 t seems likely that that there may be a mechanism for repair of DNA this translesion polymerase activity allows restart of Editorial / Mutation Research xxx (2003) xxx–xxx replication forks that have stalled due to weak poly- chemicals—adozelesin, a bulky alkylating agent, or merase ␣ Note that a “translesion” polymerase is methyl methane sulfonate (MMS), a methylating employed in this case even though no known “lesion” agent—to interfere with replication. They found that is generated when a replication fork stalls due to a these four treatments differ from each other with regard to extents of Chk1, RPA32, and H2AX phos- Replication forks naturally stall at a “replication phorylation, RPA and ␥-H2AX focus formation, and fork barrier” located downstream of the ribosomal induction of apoptosis. Their results suggest that much RNA genes in budding yeast ribosomal DNA. Weitao additional research is needed before we will fully un- et al. vide evidence that this natural stalling is derstand the variety of cellular responses to different enhanced by deletion of the gene encoding the Sgs1 DNA-damaging and replication-fork-stalling agents helicase, suggesting that Sgs1 is normally important Their results and those of Hammond et al. for restarting stalled forks. Interestingly, budding yeast also indicate that caution is needed when attempting Sgs1 is similar to the human Bloom’s and Werner’s to compare results obtained with different agents.
syndrome helicases, suggesting that some of the symp- One of the possible consequences of severe repli- toms of these syndromes may be due to inability to cation problems in metazoan cells is apoptosis. Until appropriately restart replication forks.
recently, it seemed likely that this response was con- One of the most efficient means of blocking repli- fined to multicellular organisms. However, a growing cation forks is by inhibiting the re-ligation of topoi- body of research—reviewed in this issue by Burhans somerase I-generated nicks. Pommier et al. et al. w indicates that programmed cell death thoroughly review the ways in which the resulting occurs in all organisms, prokaryotic and eukaryotic, topoisomerase I cleavage complexes can be gener- unicellular and multicellular. Furthermore, many of ated (both naturally and by topoisomerase I poisons), the phenomena of mammalian apoptosis, includ- bypassed and repaired. Cancer cells are frequently ing production of reactive oxygen species (ROS), defective in one or more of the pathways responsible activation of caspases, and inversion of cellular for repair of topoisomerase I cleavage complexes, membranes—are conserved in yeasts. Production of and this may explain the frequent hypersensitivity of ROS can be detected in both budding and fission yeasts under conditions that produce replication stress Interference with DNA replication by inhibition of What is the fate of cells in which S-phase check- polymerases (for example with aphidicolin, a specific points and repair pathways fail? As reviewed by An- inhibitor of DNA polymerases ␣, ␦ and ε), by various dreassen et al. cells move into G2 and then types of DNA damage, by depletion of dNTP pools into mitosis, where the G2 and mitotic checkpoints with hydroxyurea (HU; an inhibitor of ribonucleotide slow the cell cycle to provide an opportunity for the reductase), or by hypoxia, gives rise to distinct cel- lesions to be repaired. In mammalian cells, the G2 lular responses. The variety of such responses is and mitotic checkpoints are transient, however, and emphasized in the papers from Hammond et al. there is a possibility that the damage engendered in S-phase will persist though mitosis into the subsequent the effects on mammalian cells of inducing replica- G1-phase In many cases, mitosis with damaged tion arrest with hypoxia to the effects of inducing DNA is faulty, resulting in G1 tetraploidy. In these arrest with HU or aphidicolin. They found that HU- cases, the G1 tetraploidy checkpoint permanently ar- and aphidicolin-induced arrest were accompanied rests the cells or induces apoptosis, depending on cell by detectable DNA damage, while hypoxia-induced type Thus, the G2, mitotic and G1 tetraploidy arrest was not, and they suggest that the cellular checkpoints provide a triple backup to the S-phase responses to the damage-free replication arrest by hy- checkpoint, helping to ensure that problems arising poxia are mediated entirely by ATR, while responses during S-phase will not be propagated, because dam- to the damage-accompanied arrest induced by HU or age will be repaired or the cell carrying the damage aphidicolin are mediated by both ATR and ATM Although it is too early to predict the impact that this cells of using HU, aphidicolin, or DNA-damaging special issue of Mutation Research will have on the Editorial / Mutation Research xxx (2003) xxx–xxx fields of DNA replication, the cell cycle, and genome [6] C.H. McGowan, Running into problems: how cells cope with stability, in my opinion the high quality of each of the replicating damaged DNA, Mutat. Res. 232 (2003) 75–84.
individual articles guarantees that the issue as a whole [7] W.K. Kaufmann, T.P. Heffernan, L.M. Beaulieu, S. Doherty, A.R. Frank, Y. Zhou, M.F. Bryant, T. Zhou, D.D. Luche, N.
will make an important contribution. The high quality Nikolaishhvili-Feinberg, D.A. Simpson, M. Cordeiro-Stone, of the articles is a consequence of the thought and care Caffeine and human DNA metabolism: the magic and the of the authors and also of the helpful suggestions pro- mystery, Mutat. Res. 232 (2003) 85–102.
vided by the reviewers, whom I thank here en masse.
[8] T. Helleday, Pathways for mitotic homologous recombination Another factor contributing to the likely success of this in mammalian cells, Mutat. Res. 232 (2003) 103–115.
[9] M. Smirnova, H.L. Klein, Role of the error-free damage special issue is the fact that the authors were unusually bypass postreplication repair pathway in the maintenance of cooperative in meeting publication deadlines, and the genomic stability, Mutat. Res. 232 (2003) 117–135.
publishers were unusually efficient in preparing these [10] L. Barbour, W. Xiao, Regulation of alternative replication articles for publication. As readers can judge from the bypass pathways at stalled replication forks and its effects dates of receipt of initial and revised manuscripts, this on genome stability: a yeast model, Mutat. Res. 232 (2003)137–155.
collection of review and original research articles was [11] T. Weitao, M. Budd, J.L. Campbell, Evidence that yeast published more promptly than most single research or SGS1, DNA2, SRS2 and FOB1 interact to maintain rDNA review articles. As a result, the references contained stability, Mutat. Res. 232 (2003) 157–172.
in these review and research articles are unusually [12] Y. Pommier, C. Redon, V.A. Rao, J.A. Seiler, O. Sordet, up-to-date at the time of publication. I would like to H. Takemura, S. Antony, L. Meng, Z. Liao, G. Kohlhagen,H. Zhang, K.W. Kohn, Repair of and checkpoint response thank all of the authors, and also Peter Stambrook, the to topoisomerase I-mediated DNA damage, Mutat. Res. 232 Mutation Research Editor-in-Charge of this special is- sue, and his Secretary, Kathleen Gouge, for their coop- [13] E.M. Hammond, S.L. Green, A.J. Giaccia, Comparison of eration, encouragement and patience. The success of hypoxia-induced replication arrest with hydroxyurea- and this special issue is a consequence of their combined aphidicolin-induced arrest, Mutat. Res. 232 (2003) 205–213.
[14] J.-S. Liu, S.-R. Kuo, T. Melendy, Comparison of checkpoint responses triggered by DNA polymerase inhibition versusDNA damaging agents, Mutat. Res. 232 (2003) 215–226.
References
Ramachandran, G. D’Urso, J.A. Huberman, Apoptosis-likeyeast cell death in response to DNA damage and replication [1] J.M. Sidorova, L.L. Breeden, Precocious G1/S transitions and defects, Mutat. Res. 232 (2003) 227–243.
genomic instability: the origin connection, Mutat. Res. 232 [16] P.R. Andreassen, O.D. Lohez, R.L. Margolis, G2 and spindle assembly checkpoint adaptation, and tetraploidy [2] B.P. Duncker, G.W. Brown, Cdc7 kinases (DDKs) and arrest: implications for intrinsic and chemically induced checkpoint responses: lessons from two yeasts, Mutat. Res.
genomic instability, Mutat. Res. 232 (2003) 245–253.
[3] J.M. Kim, M. Yamada, H. Masai, Functions of mammalian Cdc7 kinase in initiation/monitoring of DNA replication and development, Mutat. Res. 232 (2003) 29–40.
[4] M.P. Longhese, M. Clerici, G. Lucchini, The S-phase checkpoint and its regulation in Saccharomyces cerevisiae, [5] M. Kai, T.S.-F. Wang, Checkpoint responses to replication stalling: inducing tolerance and preventing mutagenesis, E-mail address: [email protected]

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