C-1027-induced alterations in Epstein-Barr viral DNA replication in latently infected cultured human Raji cells: relationship to DNA damage.

This study is the first detailing drug-induced changes in EBV DNA replication intermediates (RIs). Both EBV replication inhibition and damage induction were studied in latently infected human Raji cells treated with the enediyne DNA strand-scission agent C-1027. Analysis of RIs on two-dimensional agarose gels revealed a rapid loss in the EBV bubble arc. When elongation of nascent chains was blocked by aphidicolin, this loss was inhibited, suggesting that C-1027-induced disappearance of RIs was related to maturation of preformed replication molecules in the absence of initiation of new RIs. C-1027 damage to EBV DNA was limited at concentrations where loss of the bubble arc was nearly complete, and none was detected within the replicating origin (ori P)-containing fragment, indicating that replication inhibition occurred in trans. By contrast, the non-nuclear mitochondrial genome was insensitive to replication inhibition but highly sensitive to damage induced by C-1027. C-1027-induced trans inhibition of nuclear but not mitochondrial DNA replication is consistent with a cell cycle checkpoint response to a DNA-damaging agent. EBV replication and Raji cell growth were inhibited at equivalent C-1027 doses.     

Bleomycin-induced DNA damage and repair in Xenopus laevis and Xenopus tropicalis

Microgel cell electrophoresis has been used with various species to measure breakage of DNA and DNA repair following exposure to the radiomimetic antibiotic, bleomycin. With humans, a high degree of DNA damage is considered to be predictive of cancer susceptibility. Non-isogeneic Xenopus laevis, the South African clawed toad, rarely develop spontaneous or induced cancers. Here, we investigate bleomycin-induced DNA damage and repair in splenic lymphocytes of this species to test consistency with cancer predictability. As X. laevis is pseudotetraploid in nature, while Xenopus tropicalis is diploid, we additionally explore the effect of polyploidy on DNA damage and repair in these vertebrates. The results show that higher doses of bleomycin are required to induce comparable levels of DNA damage in both Xenopus species, than in humans. X. tropicalis, the diploid, is more bleomycin-sensitive than is X. laevis. Additionally, repair rates of damaged DNA of X. laevis lymphocytes are more rapid than those of X. tropicalis, although both are hours slower than human leukocytes. While no data exist on cancer susceptibility in X. tropicalis, the results suggest greater susceptibility to cancer than X. laevis, but less than in humans. Thus, polyploidy serves as a protection against DNA damage and allows more rapid repair.     

A novel replication arrest pathway in response to DNA damage

DNA damage has been shown to regulate DNA replication both by inhibition of origin utilization, and by slowing of replication progression. We have recently reported another mechanism by which DNA damage affects replication, in which the presence of damaged DNA inhibits, in trans, the initiation of chromosomal replication. This inhibition occurs by blocking the association of the processivity clamp PCNA with undamaged chromatin. This inhibitory activity is not due to sequestration of replication factors by the damaged DNA, rather, it acts through generation of a diffusible inhibitor of PCNA loading. The activation of this pathway is independent of canonical checkpoint signaling, and, in fact, results in activation of the checkpoint. This novel pathway may therefore represent an amplification step to stop cell cycle progression in response to lower levels of DNA damage.     

DNA damage and repair in brain: relationship to aging.

The usefulness of conducting DNA damage and repair studies in a postmitotic tissue like brain is emphasized. We review studies that use brain as a tissue to test the validity of the DNA damage and repair hypothesis of aging. As far as the accumulation of age dependent DNA damage is concerned, the data appear to overwhelmingly support the hypothesis. However, attempts to demonstrate a decline in DNA repair capacity as a function of age are conflicting and equally divided. Possible reasons for this discrepancy are discussed. It is suggested that assessment of the repair capacity of neurons with respect to a specific type of damage in a specific gene might yield more definitive answers regarding the role of DNA repair potential in the aging process and as a longevity assurance system.     

Mitotic DNA damage and replication checkpoints in yeast

Studies of the genetics of G₂/M checkpoints in budding and fission yeasts have produced many of the defining concepts of checkpoint biology. Recent progress in the biochemistry of the checkpoint gene products is adding a mechanistic understanding to our models and identifying the components of the normal cell cycle machinery that are targeted by checkpoints.     

Ubiquitin-family modifications in the replication of DNA damage

The cell uses specialised Y-family DNA polymerases or damage avoidance mechanisms to replicate past damaged sites in DNA. These processes are under complex regulatory systems, which employ different types of post-translational modification. All the Y-family polymerases have ubiquitin binding domains that bind to mono-ubiquitinated PCNA to effect the switching from replicative to Y-family polymerase. Ubiquitination and de-ubiquitination of PCNA are tightly regulated. There is also evidence for another as yet unidentified ubiquitinated protein being involved in recruitment of Y-family polymerases to chromatin. Poly-ubiquitination of PCNA stimulates damage avoidance, and, at least in yeast, PCNA is SUMOylated to prevent unwanted recombination events at the replication fork. The Y-family polymerases themselves can be ubiquitinated and, in the case of DNA polymerase η, this results in the polymerase being excluded from chromatin.     

Bleomycin-induced DNA-strand damage in isolated male germ cells

DNA strand damage in isolated male germ cells (MGC) was evaluated after in vitro exposure to bleomycin (BLM), a known genotoxin. The alkaline elution technique was used to determine DNA-strand breaks. Concentration-dependent strand damage was established following exposure to bleomycin for 1 h at 37 degrees C. Exposure at 0 degrees C resulted in an increase in the frequency of strand breaks as compared to those observed at 37 degrees C. Pretreatment of cells with deferoxamine (DM), an iron-selective chelating agent, abolished the DNA damage induced by bleomycin. Isolated male germ cells responded in a predictable and reproducible manner thus supporting their use in mechanistic studies of genotoxicity.     

Tolerating DNA damage during eukaryotic chromosome replication

In eukaryotes, the evolutionarily conserved RAD6/RAD18 pathway of DNA damage tolerance overcomes unrepaired DNA lesions that interfere with the progression of replication forks, helping to ensure the completion of chromosome replication and the maintenance of genome stability in every cell cycle. This pathway uses two different strategies for damage bypass: translesion DNA synthesis, which is carried out by specialized polymerases that can replicate across the lesions, and DNA damage avoidance, a process that relies on a switch to an undamaged-DNA template for synthesis past the lesion. In this review, we summarise the current knowledge on DNA damage tolerance mechanisms mediated by RAD6/RAD18 that are used by eukaryotic cells to cope with DNA lesions during chromosome replication.     

Bleomycin-induced potentially lethal damage and its repair

The dependence of the survival of V79 Chinese hamster cells which were treated with bleomycin on a post-treatment with anisotonic phosphate-buffered saline and/or conditioned medium was examined. In qualitative similarity to the effects of these post-treatments on survival following exposure to X rays, it was found that: anisotonic saline, both hypo- and hypertonic, significantly enhanced cell killing; the degree of enhancement increased with time and temperature; and incubation in conditioned medium resulted in the rescue of cells whether or not they were challenged with hypertonic saline after treatment with bleomycin. Based upon the qualitative similarity of these findings to those which we have observed following X irradiation, and observations of others on the site of action of bleomycin in cells and the ability that it has to break DNA, we propose that both bleomycin and radiation (X rays and fast neutrons) act on similar sensitive sites which are probably contained in or close to the envelope and/or the protein matrix of the nucleus.     

Apoptosis-like yeast cell death in response to DNA damage and replication defects

In budding (Saccharomyces cerevisiae) and fission (Schizosaccharomyces pombe) yeast and other unicellular organisms, DNA damage and other stimuli can induce cell death resembling apoptosis in metazoans, including the activation of a recently discovered caspase-like molecule in budding yeast. Induction of apoptotic-like cell death in yeasts requires homologues of cell cycle checkpoint proteins that are often required for apoptosis in metazoan cells. Here, we summarize these findings and our unpublished results which show that an important component of metazoan apoptosis recently detected in budding yeast-reactive oxygen species (ROS)-can also be detected in fission yeast undergoing an apoptotic-like cell death. ROS were detected in fission and budding yeast cells bearing conditional mutations in genes encoding DNA replication initiation proteins and in fission yeast cells with mutations that deregulate cyclin-dependent kinases (CDKs). These mutations may cause DNA damage by permitting entry of cells into S phase with a reduced number of replication forks and/or passage through mitosis with incompletely replicated chromosomes. This may be relevant to the frequent requirement for elevated CDK activity in mammalian apoptosis, and to the recent discovery that the initiation protein Cdc6 is destroyed during apoptosis in mammals and in budding yeast cells exposed to lethal levels of DNA damage. Our data indicate that connections between apoptosis-like cell death and DNA replication or CDK activity are complex. Some apoptosis-like pathways require checkpoint proteins, others are inhibited by them, and others are independent of them. This complexity resembles that of apoptotic pathways in mammalian cells, which are frequently deregulated in cancer. The greater genetic tractability of yeasts should help to delineate these complex pathways and their relationships to cancer and to the effects of apoptosis-inducing drugs that inhibit DNA replication.     

Dynamic targeting of the replication machinery to sites of DNA damage

Components of the DNA replication machinery localize into discrete subnuclear foci after DNA damage, where they play requisite functions in repair processes. Here, we find that the replication factors proliferating cell nuclear antigen (PCNA) and RPAp34 dynamically exchange at these repair foci with discrete kinetics, and this behavior is distinct from kinetics during DNA replication. Posttranslational modification is hypothesized to target specific proteins for repair, and we find that accumulation and stability of PCNA at sites of damage requires monoubiquitination. Contrary to the popular notion that phosphorylation on the NH2 terminus of RPAp34 directs the protein for repair, we demonstrate that phosphorylation by DNA-dependent protein kinase enhances RPAp34 turnover at repair foci. Together, these findings support a dynamic exchange model in which multiple repair factors regulated by specific modifications have access to and rapidly turn over at sites of DNA damage.     

Reactive oxygen-mediated damage to a human DNA replication and repair protein

Ultraviolet A (UVA) makes up more than 90% of incident terrestrial ultraviolet radiation. Unlike shorter wavelength UVB, which damages DNA directly, UVA is absorbed poorly by DNA and is therefore considered to be less hazardous. Organ transplant patients treated with the immunosuppressant azathioprine frequently develop skin cancer. Their DNA contains 6-thioguanine-a base analogue that generates DNA-damaging singlet oxygen ((1)O(2)) when exposed to UVA. Here, we show that this (1)O(2) damages proliferating cell nuclear antigen (PCNA), the homotrimeric DNA polymerase sliding clamp. It causes covalent oxidative crosslinking between the PCNA subunits through a histidine residue in the intersubunit domain. Crosslinking also occurs after treatment with higher-although still moderate-doses of UVA alone or with chemical oxidants. Chronic accumulation of oxidized proteins is linked to neurodegenerative disorders and ageing. Our findings identify oxidative damage to an important DNA replication and repair protein as a previously unrecognized hazard of acute oxidative stress.     

Regulation of DNA replication by the S-phase DNA damage checkpoint

Cells slow replication in response to DNA damage. This slowing was the first DNA damage checkpoint response discovered and its study led to the discovery of the central checkpoint kinase, Ataxia Telangiectasia Mutated (ATM). Nonetheless, the manner by which the S-phase DNA damage checkpoint slows replication is still unclear. The checkpoint could slow bulk replication by inhibiting replication origin firing or slowing replication fork progression, and both mechanisms appear to be used. However, assays in various systems using different DNA damaging agents have produced conflicting results as to the relative importance of the two mechanisms. Furthermore, although progress has been made in elucidating the mechanism of origin regulation in vertebrates, the mechanism by which forks are slowed remains unknown. We review both past and present efforts towards determining how cells slow replication in response to damage and try to resolve apparent conflicts and discrepancies within the field. We propose that inhibition of origin firing is a global checkpoint mechanism that reduces overall DNA synthesis whenever the checkpoint is activated, whereas slowing of fork progression reflects a local checkpoint mechanism that only affects replisomes as they encounter DNA damage and therefore only affects overall replication rates in cases of high lesion density.     

DNA replication as a target of the DNA damage checkpoint

Faithful inheritance of the genome from mother to daughter cell requires that it is replicated accurately, in its entirety, exactly once. DNA replication not only has to have high fidelity, but also has to cope with exogenous and endogenous agents that damage DNA during the life cycle of a cell. The DNA damage checkpoint, which monitors and responds to defects in the genome, is critical for the completion of replication. The focus of this review is how DNA replication is regulated by the checkpoint response in the presence of DNA damage and fork stalling agents.     

Photo-induced hole transfer from base to sugar in DNA: relationship to primary radiation damage

This work presents the hypothesis that photo-excitation of G.+ in DNA and model systems results in the same electronic states expected from direct ionization of the sugar phosphate backbone and that these states lead to specific sugar radicals on the DNA sugar phosphate backbone. As evidence we show that visible photo-excitation of guanine cation radicals (G.+) in the dinucleoside phosphate TpdG results in high yields (about 85%) of deoxyribose sugar radicals at the C1' and C3' sites. Further, we have calculated transition energies of hole transfer from G.+ in TpdG using time-dependent density functional theory (TD-DFT) at the B3LYP/6-31G(d) level in gas phase as well as in a solvated environment. These calculations clearly predict that visible excitation of G.+ in TpdG causes transitions from only inner-shell filled molecular orbitals (MOs) to the singly occupied molecular orbital (SOMO) that effectively result in hole transfer from guanine either to the sugar phosphate backbone or to the adjacent base, thymine. The hole transfer is followed by rapid deprotonation from the sugar to form C1' and C3' radicals. These experimental and theoretical results are in agreement with our previously published experimental and theoretical results that photo-excitation of G.+ results in high yields of deoxyribose sugar radicals in DNA, guanine deoxyribonucleosides and deoxyribonucleotides. Photo-excitation of G.+ therefore provides a convenient method to produce and study sugar radicals that are expected to be formed in gamma-irradiated DNA systems unencumbered by the many other pathways involved in direct ionization.     

Subtle alterations in PCNA-partner interactions severely impair DNA replication and repair

The robustness of complex biological processes in the face of environmental and genetic perturbations is a key biological trait. However, while robustness has been extensively studied, little is known regarding the fragility of biological processes. Here, we have examined the susceptibility of DNA replication and repair processes mediated by the proliferating cell nuclear antigen (PCNA). Using protein directed evolution, biochemical, and genetic approaches, we have generated and characterized PCNA mutants with increased affinity for several key partners of the PCNA-partner network. We found that increases in PCNA-partner interaction affinities led to severe in vivo phenotypic defects. Surprisingly, such defects are much more severe than those induced by complete abolishment of the respective interactions. Thus, the subtle and tunable nature of these affinity perturbations produced different phenotypic effects than realized with traditional "on-off" analysis using gene knockouts. Our findings indicate that biological systems can be robust to one set of perturbations yet fragile to others.