Mechanisms of dealing with DNA damage-induced replication problems
ABSTRACT 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.
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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.DNA repair 05/2014; DOI:10.1016/j.dnarep.2014.04.008
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ABSTRACT: Nucleotide excision repair (NER) plays an essential role in many organisms across life domains to preserve and faithfully transmit DNA to the next generation. In humans, NER is essential to prevent DNA damage-induced mutation accumulation and cell death leading to cancer and aging. NER is a versatile DNA repair pathway that repairs many types of DNA damage which distort the DNA helix, such as those induced by solar UV light. A detailed molecular model of the NER pathway has emerged from in vitro and live cell experiments, particularly using model systems such as bacteria, yeast, and mammalian cell cultures. In recent years, the versatility of the nematode C. elegans to study DNA damage response (DDR) mechanisms including NER has become increasingly clear. In particular, C. elegans seems to be a convenient tool to study NER during the UV response in vivo, to analyze this process in the context of a developing and multicellular organism, and to perform genetic screening. Here, we will discuss current knowledge gained from the use of C. elegans to study NER and the response to UV-induced DNA damage.08/2011; 2011:542795. DOI:10.4061/2011/542795
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ABSTRACT: High-fidelity replication of DNA, and its accurate segregation to daughter cells, is critical for maintaining genome stability and suppressing cancer. DNA replication forks are stalled by many DNA lesions, activating checkpoint proteins that stabilize stalled forks. Stalled forks may eventually collapse, producing a broken DNA end. Fork restart is typically mediated by proteins initially identified by their roles in homologous recombination repair of DNA double-strand breaks (DSBs). In recent years, several proteins involved in DSB repair by non-homologous end joining (NHEJ) have been implicated in the replication stress response, including DNA-PKcs, Ku, DNA Ligase IV-XRCC4, Artemis, XLF and Metnase. It is currently unclear whether NHEJ proteins are involved in the replication stress response through indirect (signaling) roles, and/or direct roles involving DNA end joining. Additional complexity in the replication stress response centers around RPA, which undergoes significant post-translational modification after stress, and RAD52, a conserved HR protein whose role in DSB repair may have shifted to another protein in higher eukaryotes, such as BRCA2, but retained its role in fork restart. Most cancer therapeutic strategies create DNA replication stress. Thus, it is imperative to gain a better understanding of replication stress response proteins and pathways to improve cancer therapy.Journal of Molecular Cell Biology 02/2011; 3(1):4-12. DOI:10.1093/jmcb/mjq049