In metazoans, accurate replication of chromosomes is ensured by the coupling of DNA synthesis to the synthesis of histone proteins. Expression of replication-dependent histone genes is restricted to S-phase by a combination of cell cycle-regulated transcriptional and post-transcriptional control mechanisms and is linked to DNA replication by a poorly understood mechanism involving checkpoint kinases [Su, Gao, Schneider, Helt, Weiss, O'Reilly, Bohmann and Zhao (2004) EMBO J. 23, 1133–1143; Kaygun and Marzluff (2005) Nat. Struct. Mol. Biol. 12, 794–800]. Here we propose a model for the molecular mechanisms that link these two important processes within S-phase, and propose roles for multiple checkpoints in this mechanism.
Cell cycle checkpoints control the response to genotoxic stress in eukaryotes
During the evolution of eukaryotes, molecular mechanisms were established that ensure accurate DNA replication and appropriate assembly of chromatin. In S-phase, DNA replication and histone protein synthesis are finely balanced; disturbances can result in misregulation of gene expression, cell cycle arrest and chromosome instability, any one of which can result in developmental failure. Several checkpoints may be activated in S-phase in response to genotoxic stress. These checkpoints delay cell cycle progression and inhibit DNA replication by stabilizing arrested replisome components and preventing any further replication initiation from later-firing replication origins.
PIKK (phosphatidylinositol 3-kinase-like kinase) family members are key components of cell cycle checkpoints
ATM (ataxia-telangiectasia mutated) and ATR (ataxia-telangiectasia and Rad3 related) checkpoint kinases are members of the PIKK family which are activated by various forms of DNA damage. ATR is activated by aberrant DNA structures induced by UV light or replicational stress caused by DNA synthesis inhibitors, whereas ATM is mainly activated by DSBs (DNA double-strand breaks), resulting, for example, from exposure to ionizing irradiation . ATM or ATR activate Chk (cell cycle checkpoint kinase) 1 and 2 effector kinases that have partially overlapping functions. Chk1 functions include the activation of the homologous recombination repair machinery and, in metazoans, activation of the origin firing and replisome integrity checkpoint . DNA-PK (DNA-activated protein kinase) is a further member of the PIKK family required primarily for DSB repair by NHEJ (non-homologous end-joining) and telomere maintenance. DNA-PK is activated by DSBs caused, for example, by the collapse of stalled replication forks, and DNA-PK targets include components of the DNA replication/repair machinery . hSMG-1 (human SMG-1) is the most recently identified PIKK family member involved in the response to genotoxic stress. hSMG-1 is necessary for the maintenance of genomic integrity as well as for NMD (nonsense-mediated mRNA decay) as well as phosphorylating p53 and the nonsense-mediated decay factor UPF-1 (homologue of Saccharomyces cerevisiae up-frameshift mutation 1) .
Checkpoints that control metazoan histone gene expression
Activation of the p53-dependent G1 checkpoint in response to ionizing radiation in human cells results in inhibition of histone gene transcription . Replicational stress activates inter-S-phase checkpoints and causes the selective destabilization of histone mRNA, resulting in the cessation of histone protein synthesis. The destabilization of histone mRNA is attenuated by treatment with caffeine, an inhibitor of ATM and ATR, and by the inhibition of ATR signalling, implicating ATR, a key component of an intermediate S-phase checkpoint, in this response to replicational stress . The effect of the inhibition of ATR signalling is variable and transient, and complete stabilization of histone mRNA can be achieved by treatment with translation inhibitors and protein phosphatase inhibitors. This indicates that further signalling mechanisms link histone gene expression with DNA replication. We have obtained evidence for a further signalling pathway co-ordinating DNA replication with histone mRNA stability (B. Müller, J. Blackburn, C. Feijoo, X. Zhao and C. Smythe, unpublished work). Here we propose a model where different signalling pathways, controlled by activation of known checkpoint mediators or other, so far unidentified, sensors of genotoxic stress control the stability of histone mRNA (Figure 1). A key feature of this model is that various signalling molecules, specifically activated by different DNA lesions, contribute to the control of histone mRNA stability. Replicational stress-induced stalling of replication forks activates ATR. In addition, the stalled replication forks will collapse at a certain frequency, resulting in double-strand breaks capable of activating the ATM or DNA-PK pathway. Activation of multiple checkpoint mediators would be expected to contribute to the overall efficiency of histone mRNA decay in response to replicational stress (Figure 1). It is likely that the contribution of these, at least partially redundant, signalling pathways to the control of histone gene expression will differ depending on the nature and frequency of the activating DNA lesion.
Replicational stress causes rapid decay of histone mRNA via a currently poorly understood pathway, perhaps involving hSMG-1 (upper right-hand arrow)
Molecular mechanism controlling histone mRNA stability
The effectors of these signalling pathways have not been identified yet, but may be among the factors known to be involved in histone mRNA stability control. Histone mRNA stability control is critically dependent on the sequence and position of the conserved hairpin element at the 3′ end of histone mRNAs, and on translation of the mRNA . The hairpin is the binding site for the RNA-binding protein HBP/SLBP (histone hairpin-binding protein/stem–loop-binding protein) , which is cell cycle-regulated by a combination of translational up-regulation in late G1-phase and cyclin-directed protein decay in G2-phase . However, HBP/SLBP protein levels and ability to bind to RNA are not affected by replicational stress, indicating that histone mRNA destabilization is not caused by the degradation or dissociation from histone mRNA of HBP/SLBP .
The RNA helicase UPF-1 has been implicated in histone mRNA decay . UPF-1 is involved in NMD and in a related process, SMD (Staufen-mediated decay) . Inhibition of UPF-1 function reduces the efficiency of replicational stress-induced histone mRNA decay . In SMD, UPF-1 is recruited to mRNAs by binding to the RNA binding protein Staufen. Similarly, UPF-1 may be recruited to histone mRNA by direct, or indirect, protein–protein interactions with HBP/SLBP . UPF-1 is a direct target of ATR and hSMG-1 , and possibly also of DNA-PK. Furthermore, HBP/SLBP contains serine/threonine-glutamine sites that are preferentially phosphorylated by ATM, ATR, DNA-PK and hSMG-1. An attractive hypothesis is that changes in HBP/SLBP or UPF-1 protein phosphorylation, caused directly or indirectly by activation of PIKK family members in response to genotoxic stress, contribute to this interaction and ensure the control of histone mRNA stability. Subsequent degradation steps may involve other components of the NMD machinery. Alternatively, the histone-specific exonuclease 3′hExo , which also contains serine/threonine-glutamine protein phosphorylation sites, may be activated by signalling, resulting in 3′–5′ decay of histone mRNA, perhaps also involving the exosome, the major 3′–5′ exonuclease complex involved in mRNA decay .
We propose that several, at least in part, redundant signalling pathways ensure that histone gene expression is linked to DNA replication. Members of the PIKK family activated by replicational stress cause the destabilization of the histone message. The contribution of the different kinases, which are specifically activated by various DNA lesions, is dependent on the frequency with which these lesions occur. Downstream effectors of these signalling pathways may include NMD factors as well as factors involved in histone mRNA metabolism.
Cancer: A Focus Topic at Life Sciences 2007, held at SECC Glasgow, U.K., 9–12 July 2007. Edited by D. Gillespie (Beatson Institute, Glasgow, U.K.) and H.M. Wallace (Aberdeen, U.K.).
ataxia-telangiectasia and Rad3-related
cell cycle checkpoint kinase
DNA-activated protein kinase
DNA double-strand break
histone hairpin-binding protein/stem–loop-binding protein
nonsense-mediated mRNA decay
phosphatidylinositol 3-kinase-like kinase
homologue of Saccharomyces cerevisiae up-frameshift mutation 1