DNA repair mechanisms in dividing and non-dividing cells

DNA damage created by endogenous or exogenous genotoxic agents can exist in multiple forms, and if allowed to persist, can promote genome instability and directly lead to various human diseases, particularly cancer, neurological abnormalities, immunodeficiency and premature aging. To avoid such deleterious outcomes, cells have evolved an array of DNA repair pathways, which carry out what is typically a multiple-step process to resolve specific DNA lesions and maintain genome integrity. To fully appreciate the biological contributions of the different DNA repair systems, one must keep in mind the cellular context within which they operate. For example, the human body is composed of non-dividing and dividing cell types, including, in the brain, neurons and glial cells. We describe herein the molecular mechanisms of the different DNA repair pathways, and review their roles in non-dividing and dividing cells, with an eye toward how these pathways may regulate the development of neurological disease.     

UV-induced cell damage is species-specific among aquatic phagotrophic protists

The sensitivity to ultraviolet radiation (UVR, 280-400 nm) of ten species of freshwater and marine phagotrophic protists was assessed in short-term (4 h) laboratory experiments. Changes in the motility and morphology of the cells, as well as direct quantification of DNA damage, were evaluated. The net amount of cyclobutane pyrimidine dimers formed after exposure of the organisms to a weighted dose (Setlow DNA normalized at 300 nm) of 1.7 kJ m(-2) was quantified by an immunoassay using a monoclonal specific antibody directed against thymine dimers (T<>Ts). This is the first application of this method to aquatic protists. The results indicated that marine and freshwater heterotrophic nanoflagellates, representatives from the order Kinetoplastida (Bodo caudatus and Bodo saltans, respectively) accumulate significantly higher DNA damage than protists representatives of the orders Chrysomonadida, Cryptomonadida or Scuticociliatida. The high proportion of A:T bases in the unique kinetoplast DNA, may explain the higher accumulation of T<>Ts found in bodonids. Experiments made with B. saltans to study the dynamics of DNA damage accumulation in the presence of UVR and photorepairing light, indicated that the mechanisms of DNA repair in this species are very inefficient. Furthermore, the dramatic changes observed in the cell morphology of B. saltans probably compromise its recovery. Our results show that sensitivity to UVR among aquatic phagotrophic protists is species-specific and that different cell targets are affected differently among species. While DNA damage in B. saltans was accompanied by motility reduction, altered morphology, and finally mortality, this was not observed in other bodonids as well as in the other species tested.     

A damaged DNA binding protein 2 mutation disrupting interaction with proliferating-cell nuclear antigen affects DNA repair and confers proliferation advantage

In mammalian cells, Nucleotide Excision Repair (NER) plays a role in removing DNA damage induced by UV radiation. In Global Genome-NER subpathway, DDB2 protein forms a complex with DDB1 (UV-DDB), recognizing photolesions. During DNA repair, DDB2 interacts directly with PCNA through a conserved region in N-terminal tail and this interaction is important for DDB2 degradation. In this work, we sought to investigate the role of DDB2-PCNA association in DNA repair and cell proliferation after UV-induced DNA damage. To this end, stable clones expressing DDB2^Wt and DDB2^PCNA- were used. We have found that cells expressing a mutant DDB2 show inefficient photolesions removal, and a concomitant lack of binding to damaged DNA in vitro. Unexpected cellular behaviour after DNA damage, such as UV-resistance, increased cell growth and motility were found in DDB2^PCNA- stable cell clones, in which the most significant defects in cell cycle checkpoint were observed, suggesting a role in the new cellular phenotype. Based on these findings, we propose that DDB2-PCNA interaction may contribute to a correct DNA damage response for maintaining genome integrity.     

DEPTH DISTRIBUTIONS OF DNA DAMAGE IN ANTARCTIC MARINE PHYTO- AND BACTERIOPLANKTON EXPOSED TO SUMMERTIME UV RADIATION

During a survey from January to March 1998, the occurrence of UV-B radiation (UVBR)- induced DNA damage in Antarctic marine phytoplankton and bacterioplankton was investigated. Sampling was done in Ryder Bay, off the British base Rothera Station, 67°S, 68°W (British Antarctic Survey). Samples were taken regularly during the survey period at fixed depths, after which DNA damage was measured in various plankton size fractions (>10, 2–10, and 0.2–2 μm). Incident solar radiation was measured using spectroradiometry, whereas attenuation of biologically effective UVBR was studied using a DNA dosimeter. A diatom bloom was found in the bay during the research period, judging from microscopic observations and HPLC analyses of taxon-specific pigments. The high phytoplankton biomass likely caused strong attenuation of DNA effective UVBR (K_bd-eff). K_bd-eff values ranged from 0.83·m ^{− 1} at the peak of the bloom to 0.47·m ^{− 1} at the end of the season. UVBR-mediated DNA damage, as measured by cyclobutane pyrimidine dimer (CPD) abundance, was detected in all plankton size fractions. Highest levels were found in the smallest size fraction, mainly consisting of heterotrophic bacteria. Clear CPD depth profiles were found during mid-summer (January, beginning of February) with surface levels exceeding 100 CPDs per million nucleotides in the bacterioplankton fraction. At that time, melting of the continuously present shelf ice caused strong salinity gradients in the upper meters, thereby stimulating water column stabilization. At the end of February and beginning of March, this phenomenon was less pronounced or absent. At that time, DNA damage was homogeneously distributed over the first 10 m, ranging between 20 and 30 CPDs per million nucleotides for the smallest size fraction.     

Should I stay or should I go: VCP/p97-mediated chromatin extraction in the DNA damage response

The ordered assembly of DNA repair factors on chromatin has been studied in great detail, whereas we are only beginning to realize that selective extraction of proteins from chromatin plays a central role in the DNA damage response. Interestingly, the protein modifier ubiquitin not only regulates the well-documented recruitment of repair proteins, but also governs the temporally and spatially controlled extraction of proteins from DNA lesions. The facilitator of protein extraction is the ubiquitin-dependent ATPase valosin-containing protein (VCP)/p97 complex, which, through its segregase activity, directly extracts ubiquitylated proteins from chromatin. In this review, we summarize recent studies that uncovered this important role of VCP/p97 in the cellular response to genomic insults and discuss how ubiquitin regulates two intuitively counteracting activities at sites of DNA damage.     

Thallus morphology and optical characteristics affect growth and DNA damage by UV radiation in juvenile Arctic Laminaria sporophytes

Growth of young sporophytes of the brown algae Laminaria digitata, L. saccharina and L. solidungula from Spitsbergen were measured in the laboratory after being exposed for 21 days to either photosynthetically active radiation (PAR=P) or to full light spectrum (PAR + UV-A + UV-B=PAB) using of cutoff glass filters. The plants were grown at 8±2°C and 16 h light : 8 h dark cycles with 6 h additional ultraviolet radiation (UVR) exposure in the middle of the light period. Growth was measured every 10 min using growth chambers with online video measuring technique. Tissue morphology and absorption spectra were measured in untreated young sporophytes while chlorophyll (Chl) a content and DNA damage were measured in treated thalli at the end of the experiment. In all species, growth rates were significantly higher in sporophytes exposed to P alone compared to sporophytes exposed to PAB. Tissue DNA damage is dependent on thallus thickness and absorption spectra characteristics of pigments and UV-absorbing compounds. In sporophytes exposed to UVR, energy demands for repair of DNA damage and synthesis of UV-absorbing compounds for protection effectively diverts photosynthate at the expense of growth. Photosynthetic pigment was not significantly different between treatments suggesting a capacity for acclimation to moderate UVR fluence. The general growth pattern in sporophytes exposed to P alone showed an increasing growth rate from the onset of light (0500–0900 hours) to a peak at the middle of the light phase (0900–1500 hours), a decline towards the end of the light phase (1500–2100 hours) and a minimum “low” growth in the dark (2100–0500 hours) relative to growth during the entire light phase. Under PAB, different growth patterns were observed such as growth compensation at night in L. digitata, delayed growth recovery in L. saccharina and minimal but continuous growth in L. solidungula. Growth as an integrative parameter of all physiological processes showed that the effect of UVR is correlated to the depth distribution of these species.     

高等植物UV-B效应研究进展

本文概述了植物ＵＶ－Ｂ效应近年来的研究进展,ＵＶ－Ｂ对植物生理过程的影响表现为抑制细胞伸长,降低光合作用,引起植物细胞内活性氧代谢的紊乱,膜脂过氧化作用增强。植物种间、种内都存在ＵＶ－Ｂ敏感性差异。ＵＶ－Ｂ对植物ＤＮＡ的损伤主要是形成嘧啶二聚体。ＵＶ－Ｂ可诱导紫外吸收化合物的合成,积累,并对植物基因表达有重要调节作用。     

Temperature dependence of UV radiation effects in Arctic and temperate isolates of three red macrophytes

The temperature dependence of UV effects was studied for Arctic and temperate isolates of the red macrophytes Palmaria palmata, Coccotylus truncatus and Phycodrys rubens. The effects of daily repeated artificial ultraviolet B and A radiation (UVBR: 280–320?nm, UVAR: 320–400?nm) treatments were examined for all isolates at 6, 12 and 18?°C by measuring growth, optimal quantum yield of PSII (F_v/F_m) and cyclobutane-pyrimidine dimer (CPD) accumulation. Furthermore, possible ecotypic differences in UV sensitivity between Arctic and temperate isolates were evaluated. Large species-specific differences in UV sensitivity were observed for all parameters: the lower subtidal species C. truncatus and P. rubens were highly sensitive to the UV treatments, whereas P. palmata, which predominantly occurs in the upper subtidal zone, was not affected by these treatments. Only minor differences were found between Arctic and temperate isolates, suggesting that no differences in UV sensitivity have evolved in these species. Relative growth rates were temperature-dependent, whereas species-specific UV effects on growth rates were relatively independent of temperature. In contrast, the species-specific decrease in F_v/F_m and its subsequent recovery were temperature-dependent in all species. UV effects on F_v/F_m were lower at 12 and 18?°C compared with 6?°C. In addition, UV effects on F_v/F_m decreased in the course of the experiment at all temperatures, indicating acclimation to the UV treatments. CPDs accumulated during the experiment in both isolates of P. rubens, whereas CPD concentrations remained low for the other two species. CPD accumulation appeared to be independent of temperature. The results suggest that summer temperatures occurring in temperate regions facilitate repair of UV-induced damage and acclimation to UV radiation in these algae compared with Arctic temperatures. Because the differences in UV effects on F_v/F_m, growth and CPD accumulation were relatively small over a broad range of temperatures, it was concluded that the influence of temperature on UV effects is small in these species.     

Differential apoptotic pathways in human keratinocyte HaCaT cells exposed to UVB and UVC

The induction of apoptosis in keratinocytes by ultraviolet (UV)-irradiation is considered to be a protective function against skin cancer. UV-induced DNA damage is a crucial event in UVB- and UVC-mediated apoptosis. However, the differences between the UVB- and UVC-induced apoptotic pathways remain unclear. Here we examine the differential mechanisms by which UVB and UVC irradiations induce keratinocyte apoptosis using human keratinocyte HaCaT cells. Differences in the production of (6-4)photoproducts ((6-4)PPs) and cyclobutane pyrimidine dimers (CPDs) were measured following irradiation with UVB and UVC at doses causing the same extent of apoptotic cell death. In addition, main apoptotic features, such as caspase activation and its regulation, were compared between UVB- and UVC-induced apoptosis. Exposures of 500 J/m² UVB and 100 J/m² UVC resulted in apoptosis to almost the same extent. At these apoptotic doses, the amounts of both (6-4)PPs and CPDs were significantly larger in the case of UVC irradiation than UVB irradiation; in parallel, the release of cytochrome c and Smac/DIABLO and the activation of caspases-9 following UVC irradiation were greater than after UVB irradiation. Importantly, caspase-8 activation occurred only in UVB-irradiated cells. Furthermore, the activation of caspase-8 was not inhibited by caspases-9 and -3 specific tetrapeptide inhibitors, indicating that the caspase-8 cleavage is not due to feedback from activation of caspases-9 and -3. Thus, these results clearly suggest that the reason apoptosis is induced to the same extent by UVB irradiation as by UVC irradiation, despite the lower production of photoproducts in DNA by UVB irradiation, is attributable to the additional activation of the caspase-8 pathway. Thus, UVB irradiation induces apoptosis through both mitochondrial (intrinsic) and caspase-8 activation (extrinsic) pathways, while UVC induces apoptosis only via the intrinsic pathway.