Mechanisms of Dealing with DNA Damage-Induced Replication Problems
Department of Cell Biology & Genetics, Cancer Genomics Center, Rotterdam, The Netherlands. Cell biochemistry and biophysics
(Impact Factor: 1.68).
12/2008; 53(1):17-31. DOI: 10.1007/s12013-008-9039-y
During every S phase, cells need to duplicate their genomes so that both daughter cells inherit complete copies of genetic information. Given the large size of mammalian genomes and the required precision of DNA replication, genome duplication requires highly fine-tuned corrective and quality control processes. A major threat to the accuracy and efficiency of DNA synthesis is the presence of DNA lesions, caused by both endogenous and exogenous damaging agents. Replicative DNA polymerases, which carry out the bulk of DNA synthesis, evolved to do their job extremely precisely and efficiently. However, they are unable to use damaged DNA as a template and, consequently, are stopped at most DNA lesions. Failure to restart such stalled replication forks can result in major chromosomal aberrations and lead to cell dysfunction or death. Therefore, a well-coordinated response to replication perturbation is essential for cell survival and fitness. Here we review how this response involves activating checkpoint signaling and the use of specialized pathways promoting replication restart. Checkpoint signaling adjusts cell cycle progression to the emergency situation and thus gives cells more time to deal with the damage. Replication restart is mediated by two pathways. Homologous recombination uses homologous DNA sequence to repair or bypass the lesion and is therefore mainly error free. Error-prone translesion synthesis employs specialized, low fidelity polymerases to bypass the damage.
Available from: Greg G Oakley
- "RPA bound to ssDNA at stalled forks recruits and activates ATR through a Rad17-RFC, 9-1-1, MRN, and TopBP1-dependent pathway. ATR phosphorylates/activates Chk1 which signals downstream factors that stabilize and repair forks, arrest active forks, and stimulate dormant origin firing to complete replication adjacent to stalled forks  . The RPA32 subunit of RPA is phosphorylated on Ser23 and Ser29 by CDK cyclically during the cell cycle, and in response to replication stress on Ser33 by ATR, and Ser4/Ser8, Ser12, and Thr21 by one or more PIKKs depending on the replication stress agent [13,20,32–36]. "
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ABSTRACT: Genotoxins and other factors cause replication stress that activate the DNA damage response (DDR), comprising checkpoint and repair systems. The DDR suppresses cancer by promoting genome stability, and it regulates tumor resistance to chemo- and radiotherapy. Three members of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, ATM, ATR, and DNA-PK, are important DDR proteins. A key PIKK target is replication protein A (RPA), which binds single-stranded DNA and functions in DNA replication, DNA repair, and checkpoint signaling. An early response to replication stress is ATR activation, which occurs when RPA accumulates on ssDNA. Activated ATR phosphorylates many targets, including the RPA32 subunit of RPA, leading to Chk1 activation and replication arrest. DNA-PK also phosphorylates RPA32 in response to replication stress, and we demonstrate that cells with DNA-PK defects, or lacking RPA32 Ser4/Ser8 targeted by DNA-PK, confer similar phenotypes, including defective replication checkpoint arrest, hyper-recombination, premature replication fork restart, failure to block late origin firing, and increased mitotic catastrophe. We present evidence that hyper-recombination in these mutants is ATM-dependent, but the other defects are ATM-independent. These results indicate that DNA-PK and ATR signaling through RPA32 plays a critical role in promoting genome stability and cell survival in response to replication stress.
Available from: Aparna Gorthi
- "The ATR protein kinase is a member of the phosphoinositide 3 kinase (PIKK) family, and is the orthologue of Saccharomyces cerevisiae Mec1. Activation of ATR by replication-blocking DNA damage elicits a pleiotropic signal transduction pathway that includes numerous transducer and effector proteins . "
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ABSTRACT: DNA replication fork stalling or collapse that arises from endogenous damage poses a serious threat to genome stability, but cells invoke an intricate signaling cascade referred to as the DNA damage response (DDR) to prevent such damage. The gene product ataxia telangiectasia and Rad3-related (ATR) responds primarily to replication stress by regulating cell cycle checkpoint control, yet it's role in DNA repair, particularly homologous recombination (HR), remains unclear. This is of particular interest since HR is one way in which replication restart can occur in the presence of a stalled or collapsed fork. Hypomorphic mutations in human ATR cause the rare autosomal-recessive disease Seckel syndrome, and complete loss of Atr in mice leads to embryonic lethality. We recently adapted the in vivo murine pink-eyed unstable (pun) assay for measuring HR frequency to be able to investigate the role of essential genes on HR using a conditional Cre/loxP system. Our system allows for the unique opportunity to test the effect of ATR loss on HR in somatic cells under physiological conditions. Using this system, we provide evidence that retinal pigment epithelium (RPE) cells lacking ATR have decreased density with abnormal morphology, a decreased frequency of HR and an increased level of chromosomal damage.
Available from: PubMed Central
- "A detailed description of the ATR-signaling pathway is beyond the scope of this review and can be found in other reviews (i.e., ); however, it is important to mention that replication checkpoint signaling is stimulated by formation of extensive RPA-coated ssDNA regions, an intermediate produced at stalled forks, and involves subsequent recruitment and modification of various components of the signaling cascade. A large body of evidence has revealed that this complex pathway, once activated, regulates origin firing, cell cycle arrest, stabilization, and resumption of stalled forks [39–41]. "
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ABSTRACT: Common fragile sites (CFS) are heritable nonrandomly distributed loci on human chromosomes that exhibit an increased frequency of chromosomal breakage under conditions of replication stress. They are considered the preferential targets for high genomic instability from the earliest stages of human cancer development, and increased chromosome instability at these loci has been observed following replication stress in a subset of human genetic diseases. Despite their biological and medical relevance, the molecular basis of CFS fragility in vivo has not been fully elucidated. At present, different models have been proposed to explain how instability at CFS arises and multiple factors seem to contribute to their instability. However, all these models involve DNA replication and suggest that replication fork stalling along CFS during DNA synthesis is a very frequent event. Consistent with this, the maintenance of CFS stability relies on the ATR-dependent checkpoint, together with a number of proteins promoting the recovery of stalled replication forks. In this review, we discuss mainly the possible causes that threaten the integrity of CFS in the light of new findings, paying particular attention to the role of the S-phase checkpoint.
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