Friday 26 September 2003
The repair of DNA lesions that occur endogenously or in response to diverse genotoxic stresses is indispensable for genome integrity.
DNA lesions activate checkpoint pathways that regulate specific DNA-repair mechanisms in the different phases of the cell cycle. Checkpoint-arrested cells resume cell-cycle progression once damage has been repaired, whereas cells with unrepairable DNA lesions undergo permanent cell-cycle arrest or apoptosis.
The cell cycle has its own internal controls, called checkpoints. There are two main checkpoints, one at the G1/S transition and another at G2/M.
The S phase is the point of no return in the cell cycle, and before a cell makes the final commitment to replicate, the G1/S checkpoint checks for DNA damage.
If DNA damage is present, the DNA repair machinery and mechanisms that arrest the cell cycle are put in motion. The delay in cell-cycle progression provides the time needed for DNA repair; if the damage is not repairable, apoptotic pathways are activated to kill the cell.
Thus, the G1/S checkpoint prevents the replication of cells that have defects in DNA, which would be perpetuated as mutations or chromosomal breaks in the progeny of the cell. DNA damaged after its replication can still be repaired as long as the chromatids have not separated.
The G2/M checkpoint monitors the completion of DNA replication and checks whether the cell can safely initiate mitosis and separate sister chromatids. This checkpoint is particularly important in cells exposed to ionizing radiation.
Cells damaged by ionizing radiation activate the G2/M checkpoint and arrest in G2; defects in this checkpoint give rise to chromosomal abnormalities.
DNA damage sensing
To function properly, cell-cycle checkpoints require sensors of DNA damage, signal transducers, and effector molecules. The sensors and transducers of DNA damage appear to be similar for the G1/S and G2/M checkpoints.
They include, as sensors, proteins of the RAD family and ataxia telangiectasia mutated (ATM) and as transducers, the CHK kinase families. The checkpoint effector molecules differ, depending on the cell-cycle stage at which they act.
In the G1/S checkpoint, cell-cycle arrest is mostly mediated through p53, which induces the cell-cycle inhibitor p21. Arrest of the cell cycle by the G2/M checkpoint involves both p53-dependent and independent mechanisms.
Defect in cell-cycle checkpoint components is a major cause of genetic instability in cancer cells.
The cell cycle proceeds by a defined sequence of events where late events depend upon completion of early events. The aim of the dependency of events is to distribute complete and accurate replicas of the genome to daughter cells. To monitor this dependency, cells are equipped with the checkpoints that are set at various stages of the cell cycle.
When cells have DNA damages that have to be repaired, cells activate DNA damage checkpoint that arrests cell cycle. According to the cell cycle stages, DNA damage checkpoints are classified into at least 3 checkpoints: G1/S (G1) checkpoint, intra-S phase checkpoint, and G2/M checkpoint.
Upon perturbation of DNA replication by drugs that interfere with DNA synthesis, DNA lesions, or obstacles on DNA, cells activate DNA replication checkpoint that arrests cell cycle at G2/M transition until DNA replication is complete. There are more checkpoints such as Spindle checkpoint and Morphogenesis checkpoint.
The spindle checkpoint arrests cell cycle at M phase until all chromosomes are aligned on spindle. This checkpoint is very important for equal distribution of chromosomes. Morphogenesis checkpoint detects abnormality in cytoskeleton and arrests cell cycle at G2/M transition.
DNA replication and chromosome distribution are indispensable events in the cell cycle control. Cells must accurately copy their chromosomes, and through the process of mitosis, segregate them to daughter cells.
The checkpoints are surveillance mechanism and quality control of the genome to maintain genomic integrity. Checkpoint failure often causes mutations and genomic arrangements resulting in genetic instability.
Genetic instability is a major factor of birth defects and in the development of many diseases, most notably cancer. Therefore, checkpoint studies are very important for understanding mechanisms of genome maintenance as they have direct impact on the ontogeny of birth defects and the cancer biology.
DNA maintenance checkpoint
Accurate duplication of eukaryotic genome is a challenging task, given that environment of cell growth and division is rarely ideal. Cells are constantly under the stress of intrinsic and extrinsic agents that cause DNA damage or interference with DNA replication.
To cope with these assaults, cells are equipped with DNA maintenance checkpoints 3 to arrest cell cycle and facilitate DNA repair pathways. DNA maintenance checkpoints include (a) the DNA damage checkpoints that recognize and respond to DNA damage, and (b) the DNA replication checkpoint that monitors the fidelity of copying DNA.
- DNA damage checkpoint
DNA damage checkpoints ensure the fidelity of genetic information both by arresting cell cycle progression and facilitating DNA repair pathways. Studies on many different species have uncovered a network of proteins that form the DNA damage checkpoints. Central to this network are protein kinases of ATM/ATR family known as Tel1/Mec1 in budding yeast and Tel1/Rad3 in fission yeast. These kinases sense DNA damages and phosphorylate number of proteins that regulate cell cycle progression and DNA repair pathways.
- DNA replication checkpoint
Accurate replication of the millions or billions of DNA base pairs in a eukaryotic genome is a remarkable achievement. This accomplishment is even more astonishing when one considers for DNA synthesis are rarely ideal. Damaged template, protein complexes bound to DNA, and poor supply of dNTPs are among the many obstacles that must be overcome to replicate genome. All of these situations can stall replication forks.
Stalled forks pose grave threats to genome integrity because they can rearrange, break, or collapse through disassembly of the replication complex. The pathways that respond to replication stress are signal transduction pathways that are conserved across evolution.
Atop the pathways are also ATM/ATR family kinases. These kinases together with a trimeric checkpoint clamp (termed 9-1-1 complex) and five-subunit checkpoint clamp loader (Rad17-RFC2-RFC3-RFC4-RFC5) senses stalled replication forks and transmit a checkpoint signal.
One of major functions of replication checkpoint is to prevent the onset of mitosis by regulating mitotic control proteins such as Cdc25. But perhaps the most important activity of replication checkpoint is to stabilize and protect replication forks.
The protein kinase Cds1 (human Chk2 homolog; in human, Chk1 is a functional Cds1 homolog) is a critical effector of the replication checkpoint in the fission yeast Schizosaccharomyces pombe.
Cds1 is required to prevent stabilization of replication fork in cells treated with hydroxyurea (HU), a ribonucleotide reductase inhibitor that stalls replication by depleting dNTPs. In the budding yeast Saccharomyces cerevisiae, a failure to activate Rad53 (Chk2 homolog) is associated with collapse and regression of replication forks and gross chromosomal rearrangements in cells treated with HU.
Replication fork protection complex (FPC)
The DNA replication checkpoint stabilizes replication forks that have stalled at DNA adducts and other lesions that block DNA polymerases. In the absence of DNA replication checkpoint, stalled forks are thought to collapse, creating strand break that threatens genome stability and cell viability.
Therefore, discovering how cells cope with aberrant replication forks is essential for understanding mechanisms of genome maintenance. The Chk1 and Chk2/Cds1 checkpoint kinases, which are key mediators of DNA damage and DNA replication checkpoints, are thought to be involved in cancer development.
We found the Swi1 protein is required for survival of replication fork arrest and effective activation of Chk2 kinase in fission yeast. Swi1 forms tight complex with Swi3 protein and moves with replication forks.
Swi1-Swi3 complex is also important for proficient DNA replication even in the absence of agents that cause genotoxic stress, creating single-strand DNA gaps at replication forks.
These results led to propose Swi1-Swi3 define a replication fork protection complex (FPC) that stabilizes replication forks in a configuration that is recognized by replication checkpoint sensors.
Interestingly, Tof1 protein (Budding yeast Swi1 homolog) has been reported to have similar functions. Tof1 is also involved in Rad53 (Chk2 homolog) activation and travels with replication fork.
Tof1 is needed to restrain fork progression when DNA synthesis is inhibited by HU indicating that Tof1 is required for coordination of DNA synthesis and replisome (replication machinery) movement.
FPC may be conserved across evolution
Swi1 and Tof1 belong to a large protein family that was first defined by metazoan Tim1 (Timeless). Drosophila melanogaster and mammalian Tim1s are implicated in circadian rhythmic oscillation, whereas the Caenorhabditis elegans Tim1 is required for proper chromosome cohesion and segregation.
All species listed above have Swi3 homolgs in their genomes suggesting that Swi1-Swi3 complex may be conserved amongst eukaryotes. It will be interesting to determine whether these conserved complexes are involved in DNA replication and maintenance of genome integrity.
G1-S checkpoint (S-phase checkpoint)
cell cycle checkpoint kinases
Branzei D, Foiani M. Regulation of DNA repair throughout the cell cycle. Nat Rev Mol Cell Biol. 2008 Apr;9(4):297-308. PMID: #18285803#
Yang J, Xu ZP, Huang Y, Hamrick HE, Duerksen-Hughes PJ, Yu YN. ATM and ATR: sensing DNA damage. World J Gastroenterol. 2004 Jan 15;10(2):155-60. PMID: #14716813# (Free)
Eastman A. Cell cycle checkpoints and their impact on anticancer therapeutic strategies. J Cell Biochem. 2004 Feb 1;91(2):223-31. PMID: #14743382#
Koundrioukoff S, Polo S, Almouzni G. Interplay between chromatin and cell cycle checkpoints in the context of ATR/ATM-dependent checkpoints. DNA Repair (Amst). 2004 Aug-Sep;3(8-9):969-78. PMID: #15279783#
Cobb JA, Shimada K, Gasser SM. Redundancy, insult-specific sensors and thresholds: unlocking the S-phase checkpoint response. Curr Opin Genet Dev. 2004 Jun;14(3):292-300. PMID: #15172673#
Lisby M, Rothstein R. DNA damage checkpoint and repair centers. Curr Opin Cell Biol. 2004 Jun;16(3):328-34. PMID: #15145359#
Millband DN, Campbell L, Hardwick KG. The awesome power of multiple model systems : interpreting the complex nature of spindle checkpoint signaling. Trends Cell Biol. 2002 May ;12(5):205-9. PMID : #12062159#
Pearce AK, Humphrey TC. Integrating stress-response and cell-cycle checkpoint pathways. Trends Cell Biol. 2001 Oct;11(10):426-33. PMID: #11567876#
O’Connell MJ, Walworth NC, Carr AM. The G2-phase DNA-damage checkpoint. Trends Cell Biol. 2000 Jul;10(7):296-303. PMID: #10856933#
Zhou, B.B. and Elledge, S.J. The DNA damage response: putting checkpoints in perspective. Nature 408: 433-439, 2000
Hartwell, L. H. & Weinert, T. A. Checkpoints: controls that ensure the order of cell cycle events. Science 246, 629-34 (1989).
Russell, P. Checkpoints on the road to mitosis. Trends Biochem Sci 23, 399-402 (1998).
Nyberg, K. A., Michelson, R. J., Putnam, C. W. & Weinert, T. A. TOWARD MAINTAINING THE GENOME: DNA Damage and Replication Checkpoints. Annu Rev Genet 36, 617-56 (2002).
McGowan, C. H. & Russell, P. The DNA damage response: sensing and signaling. Curr Opin Cell Biol 16, 629-33 (2004).
McGlynn, P. & Lloyd, R. G. Recombinational repair and restart of damaged replication forks. Nat Rev Mol Cell Biol 3, 859-70 (2002).
Zhou, B. B. & Elledge, S. J. The DNA damage response: putting checkpoints in perspective. Nature 408, 433-9 (2000).
Osborn, A. J., Elledge, S. J. & Zou, L. Checking on the fork: the DNA-replication stress-response pathway. Trends Cell Biol 12, 509-16 (2002).
Boddy, M. N. & Russell, P. DNA replication checkpoint. Curr. Biol. 11, R953-R956 (2001).
Boddy, M. N., Furnari, B., Mondesert, O. & Russell, P. Replication checkpoint enforced by kinases Cds1 and Chk1. Science 280, 909-912 (1998).
Lindsay, H. D. et al. S-phase-specific activation of Cds1 kinase defines a subpathway of the checkpoint response in Schizosaccharomyces pombe. Genes Dev 12, 382-95 (1998).
Noguchi, E., Noguchi, C., Du, L. L. & Russell, P. Swi1 prevents replication fork collapse and controls checkpoint kinase Cds1. Mol Cell Biol 23, 7861-74 (2003).
Kolodner, R. D., Putnam, C. D. & Myung, K. Maintenance of genome stability in Saccharomyces cerevisiae. Science 297, 552-7 (2002).
Lopes, M. et al. The DNA replication checkpoint response stabilizes stalled replication forks. Nature 412, 557-61 (2001).
Sogo, J. M., Lopes, M. & Foiani, M. Fork reversal and ssDNA accumulation at stalled replication forks owing to checkpoint defects. Science 297, 599-602 (2002).
Tercero, J. A. & Diffley, J. F. Regulation of DNA replication fork progression through damaged DNA by the Mec1/Rad53 checkpoint. Nature 412, 553-7 (2001).
Bartek, J. & Lukas, J. Chk1 and Chk2 kinases in checkpoint control and cancer. Cancer Cell 3, 421-9 (2003).
Noguchi, E., Noguchi, C., McDonald, W. H., Yates, J. R., 3rd & Russell, P. Swi1 and Swi3 are components of a replication fork protection complex in fission yeast. Mol Cell Biol 24, 8342-55 (2004).
Foss, E. J. Tof1p regulates DNA damage responses during S phase in Saccharomyces cerevisiae. Genetics 157, 567-77 (2001).
Katou, Y. et al. S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex. Nature 424, 1078-83 (2003).
Chan, R. C. et al. Chromosome cohesion is regulated by a clock gene paralogue TIM-1. Nature 424, 1002-9 (2003).
Dalgaard, J. Z. & Klar, A. J. swi1 and swi3 perform imprinting, pausing, and termination of DNA replication in S. pombe. Cell 102, 745-51 (2000).
Barnes, J. W. et al. Requirement of mammalian Timeless for circadian rhythmicity. Science 302, 439-42 (2003).