The influence of heterochromatin on DNA double strand break repair: Getting the strong, silent type to relax

DNA non-homologous end-joining (NHEJ) and homologous recombination (HR) represent the major DNA double strand break (DSB) pathways in mammalian cells, whilst ataxia telangiectasia mutated (ATM) lies at the core of the DSB signalling response. ATM signalling plays a major role in modifying chromatin structure in the vicinity of the DSB and increasing evidence suggests that this function influences the DSB rejoining process. DSBs have long been known to be repaired with two (or more) component kinetics. The majority (～85%) of DSBs are repaired with fast kinetics in a predominantly ATM-independent manner. In contrast, ～15% of radiation-induced DSBs are repaired with markedly slower kinetics via a process that requires ATM and those mediator proteins, such as MDC1 or 53BP1, that accumulate at ionising radiation induced foci (IRIF). DSBs repaired with slow kinetics predominantly localise to the periphery of genomic heterochromatin (HC). Indeed, there is mounting evidence that chromatin complexity and not damage complexity confers slow DSB repair kinetics. ATM's role in HC-DSB repair involves the direct phosphorylation of KAP-1, a key HC formation factor. KAP-1 phosphorylation (pKAP-1) arises in both a pan-nuclear and a focal manner after radiation and ATM-dependent pKAP-1 is essential for DSB repair within HC regions. Mediator proteins such as 53BP1, which are also essential for HC-DSB repair, are expendable for pan-nuclear pKAP-1 whilst being essential for pKAP-1 formation at IRIF. Data suggests that the essential function of the mediator proteins is to promote the retention of activated ATM at DSBs, concentrating the phosphorylation of KAP-1 at HC DSBs. DSBs arising in G2 phase are also repaired with fast and slow kinetics but, in contrast to G0/G1 where they all DSBs are repaired by NHEJ, the slow component of DSB repair in G2 phase represents an HR process involving the Artemis endonuclease. Results suggest that whilst NHEJ repairs the majority of DSBs in G2 phase, Artemis-dependent HR uniquely repairs HC DSBs. Collectively, these recent studies highlight not only how chromatin complexity influences the factors required for DSB repair but also the pathway choice.     

Host-Mediated Repair of Discontinuities in DNA From T4 Bacteriophage

Discontinuities of T4 DNA which are caused by excision of UV-damaged areas, by decay of (32)P atoms, or which are present in DNA from rII(-)lig(am) (-) phage produced in a host nonpermissive for amber mutants are all repaired by bacterial enzymes after infection in the presence of chloramphenicol. Escherichia coli DNA polymerase I participates in the host-mediated repair, but an approximately 20-fold variation in the levels of host polynucleotide ligase does not affect either the kinetics or the extent of repair observed. Upon removal of chloramphenicol, host-repaired DNA from UV-irradiated phage undergoes a secondary cycle of breakage, which ultimately results in solubilization of most of the phage DNA. If the cells are co-infected with nonirradiated helper phage, the secondary breaks are repaired and the continuity of the polynucleotide chain is restored. The close coincidence in the extent of primary and secondary breakage suggests that phage-coded enzymes recognize and excise areas improperly repaired by the host. In contrast to host-mediated repair, repair mediated by rescuing phage probably restored functionality to the damaged DNA.     

Cell cycle progression in denV-transfected murine fibroblasts exposed to ultraviolet radiation.

Repair-proficient murine fibroblasts transfected with the denV gene of bacteriophage T4 repaired 70-80% of pyrimidine dimers within 24 h after exposure to 150 J/m2 ultraviolet radiation (UVR) from an FS-40 sunlamp. Under the same conditions, control cells repaired only about 20% of UVR-induced pyrimidine dimers. After UVR exposure, both control and denV-transfected cells exhibited some degree of DNA-synthesis inhibition, as determined by flow cytometric analysis of cell-cycle kinetics in propidium iodide-stained cells. DenV-transfected cells had a longer and more profound S phase arrest than control cells, but both control and denV-transfected cells had largely recovered from UVR effects on cell-cycle kinetics by 48 h after UVR exposure. Inhibition of DNA synthesis by UVR was also measured by determining post-UVR incorporation of bromodeoxyuridine (BrdU). The amount of BrdU incorporated was quantitated by determining with flow cytometry the quenching of Hoechst dye 33342 by BrdU incorporated in cellular DNA. DenV-transfected cells showed more marked inhibition of BrdU incorporation after low fluences of UVR than control cells. Differences between denV-transfected and control cells in cell-cycle kinetics following UVR exposure may be related to differences in mechanisms of repair when excision repair of pyrimidine dimers is initiated by endonuclease V instead of cellular repair enzymes.     

The efficiency of DNA strand-break repair in two fibrosarcoma tumors and in normal tissues of mice irradiated in vivo with X rays

We have used alkaline elution to study the repair of X-ray-induced DNA strand breaks in vivo in two fibrosarcoma tumors and in several normal mouse tissues after whole-body irradiation of mice with 10-12.5 Gy of X rays. Both tumors were found to repair damage significantly faster and to a greater extent than any of the normal tissues, so that by 2 hr after irradiation the level of damage in both tumors was indistinguishable from unirradiated control values. Of the normal tissues studied, liver repaired the fastest. The kinetics for the other normal tissues were essentially the same, showing an appreciable level (7-16%) of unrepaired lesions still evident after 2 hr. Even as late as 12 hr there was a significant amount of residual damage in some tissues, with testes and spleen showing the greatest level (ca. 15%). The repair kinetics for each tissue were not appropriately described by a sum of two exponentials. In contrast, previously reported data for many homogeneous mammalian cell systems in vitro and for some tissues in vivo have shown biphasic repair kinetics. This difference may be related to heterogeneity of both cell type and environment within the tissue populations used in the investigation. The faster repair of DNA strand breaks by tumor cells relative to cells from normal tissues was not readily explainable in terms of such radiobiological parameters as overall tissue oxygenation or sulfhydryl content. Rather, it appears that the degree of differentiation of the cells within the tissue population may be a major determinant of repair proficiency. Based on a model incorporating a competition between repair and fixation of sublethal lesions, these data are consistent with the idea that tumor cells may have a repair, and hence survival, advantage over normal cells in response to ionizing radiation.     

Decreased repair of radiation-induced DNA double-strand breaks with cellular differentiation.

Although the majority of mammalian cells in situ are terminally differentiated, most DNA repair studies have used proliferating cells. In an attempt to understand better the relationship between differentiation and DNA repair, we have used the murine 3T3-T proadipocyte cell line. In this model system, proliferating (stem) cells undergo growth arrest (GD cells) and subsequently terminally differentiate into adipocytes when exposed to media containing platelet-depleted human plasma. Pulsed-field gel electrophoresis was used to evaluate the induction and repair of DNA double-strand breaks (DSBs) after ionizing radiation. The levels of radiation-induced DSBs in GD and terminally differentiated cells were similar, but in both cases greater than those found in stem cells at each radiation dose tested (0 to 40 Gy); these differences appear to be due to growth arrest in G1 phase. DNA DSBs were repaired with biphasic kinetics for each cell type. For terminally differentiated cells 25% of DNA DSBs remained unrejoined compared with < 10% for GD and stem cells after a repair time of 4 h. These data indicate that terminal differentiation of 3T3-T cells is associated with a reduction in the repair of ionizing radiation-induced DNA DSBs.     

Effects of temperature on the photoreactivation of ultraviolet-b–induced dna damage in Palmaria palmata (Rhodophyta)

The accumulation of DNA damage (thymine dimers and 6-4 photoproducts) induced by ultraviolet-B radiation was studied in Palmaria palmata (L.) O. Kuntze under different light and temperature conditions, using specific monoclonal antibodies and subsequent chemiluminescent detection. Both types of damage were repaired much faster under ultraviolet-A radiation (UVAR) plus photosynthetically active radiation (PAR) than in darkness, which indicates photoreactivating activity. At 12° C, all thymine dimers were repaired after 2 h irradiation with UVAR plus PAR, whereas 6-4 photoproducts were almost completely repaired after 4 h. After 19 h of darkness, almost complete repair of 6-4 photoproducts was found, and 67% of the thymine dimers were repaired. In a second set of experiments, repair of DNA damage under UVAR plus PAR was compared at three different temperatures (0, 12, and 25° C). Again, thymine dimers were repaired faster than 6-4 photoproducts at all three temperatures. At 0° C, significant repair of thymine dimers was found but not of 6-4 photoproducts. Significant repair of both thymine dimers and 6-4 photoproducts occurred at 12 and 25° C. Optimal repair efficiency was found at 25° C for thymine dimers but at 12° C for 6-4 photoproducts, which suggests that the two photorepair processes have different temperature characteristics.     

Kinetics of DNA Repair in Ultraviolet-Irradiated and N-acetoxy-2-acetylaminofluorene-Treated Mammalian Cells

Repair kinetics after saturating doses of ultraviolet radiation (UV), N-acetoxy-2-acetylaminofluorene (AAAF), and combinations of both agents were studied in human fibroblasts proficient and deficient in excision repair, and in Chinese hamster cells (V-79) deficient in excision repair. Three techniques were used: unscheduled DNA synthesis, photolysis of DNA repaired in the presence of bromodeoxyuridine (BrdUrd), and measurements of sites sensitive to a UV-endonuclease. The repair rate appears to be approximately constant during the first few hours after treatment. Later there is a decrease with time; the magnitude of the decrease depends on the cell line. Our data show that the decrease in repair observed in repair-deficient cells treated with combinations of both agents as compared to separate treatments is due neither to the cytotoxic effects of the agents used, nor to a shutoff of the repair system by the high concentrations of AAAF employed.     

X-irradiation of cells on glass slides has a dose doubling impact

Immunofluorescence detection of gammaH2AX foci is a widely used tool to quantify the induction and repair of DNA double-strand breaks (DSBs) induced by ionising radiation. We observed that X-irradiation of mammalian cells exposed on glass slides induced twofold higher foci numbers compared to irradiation with gamma-rays. Here, we show that the excess gammaH2AX foci after X-irradiation are produced from secondary radiation particles generated from the irradiation of glass slides. Both 120 kV X-rays and (137)Cs gamma-rays induce approximately 20 gammaH2AX foci per Gy in cells growing on thin ( approximately 2 microm) plastic foils immersed in water. The same yield is obtained following gamma-irradiation of cells growing on glass slides. However, 120 kV X-rays produce approximately 40 gammaH2AX foci per Gy in cells growing on glass, twofold greater than obtained using cells irradiated on plastic surfaces. The same increase in gammaH2AX foci number is obtained if the plastic foil on which the cells are grown is irradiated on a glass slide. Thus, the physical proximity to the glass material and not morphological differences of cells growing on different surfaces accounts for the excess gammaH2AX foci. The increase in foci number depends on the energy and is considerably smaller for 25 kV relative to 120 kV X-rays, a finding which can be explained by known physical properties of radiation. The kinetics for the loss of foci, which is taken to represent the rate of DSB repair, as well as the Artemis dependent repair fraction, was similar following X- or gamma-irradiation, demonstrating that DSBs induced by this range of treatments are repaired in an identical manner.     

Repair of DNA damage induced by accelerated heavy ions in mammalian cells proficient and deficient in the non-homologous end-joining pathway

Human and rodent cells proficient and deficient in non-homologous end joining (NHEJ) were irradiated with X rays, 70 keV/microm carbon ions, and 200 keV/microm iron ions, and the biological effects on these cells were compared. For wild-type CHO and normal human fibroblast (HFL III) cells, exposure to iron ions yielded the lowest cell survival, followed by carbon ions and then X rays. NHEJ-deficient xrs6 (a Ku80 mutant of CHO) and 180BR human fibroblast (DNA ligase IV mutant) cells showed similar cell survival for X and carbon-ion irradiation (RBE = approximately 1.0). This phenotype is likely to result from a defective NHEJ protein because xrs6-hamKu80 cells (xrs6 cells corrected with the wild-type KU80 gene) exhibited the wild-type response. At doses higher than 1 Gy, NHEJ-defective cells showed a lower level of survival with iron ions than with carbon ions or X rays, possibly due to inactivation of a radioresistant subpopulation. The G(1) premature chromosome condensation (PCC) assay with HFL III cells revealed LET-dependent impairment of repair of chromosome breaks. Additionally, iron-ion radiation induced non-repairable chromosome breaks not observed with carbon ions or X rays. PCC studies with 180BR cells indicated that the repair kinetics after exposure to carbon and iron ions behaved similarly for the first 6 h, but after 24 h the curve for carbon ions approached that for X rays, while the curve for iron ions remained high. These chromosome data reflect the existence of a slow NHEJ repair phase and severe biological damage induced by iron ions. The auto-phosphorylation of DNA-dependent protein kinase catalytic subunits (DNA-PKcs), an essential NHEJ step, was delayed significantly by high-LET carbon- and iron-ion radiation compared to X rays. This delay was further emphasized in NHEJ-defective 180BR cells. Our results indicate that high-LET radiation induces complex DNA damage that is not easily repaired or is not repaired by NHEJ even at low radiation doses such as 2 Gy.     

Overexpression of human Ku70/Ku80 in rat cells resulting in reduced DSB repair capacity with appropriate increase in cell radiosensitivity but with no effect on cell recovery.

The effect of an overexpression of human Ku70/80 was studied using cells of the rat cell lines Rat-1 and R7080, the latter being transfected with the human cDNAs for Ku70 and Ku80. The overexpression was found to result in a 20% reduction of the DNA-PK activity. The kinetics of DSB repair, which was studied after exposure of the cells to 30 Gy of X rays, was biphasic and had identical half-times for Rat-1 and R7080 cells (tfast = 7 min and tslow = 135 min). However, there was a significant difference between the cell lines in the fractions of DSBs repaired with slow and fast kinetics. In R7080 cells, about twice as many DSBs were repaired with slow kinetics compared to Rat-1 cells (34% compared to 16%). A similar difference was found in the number of residual DSBs (3.6% compared to 2.0%). R7080 cells also showed a reduced capacity to repair chromosome damage as detected by the PCC technique. Concerning cell killing, R7080 cells were clearly more radiosensitive than Rat-1 cells (D0.1 = 6.4 compared to 10.5 Gy), and this increase in sensitivity correlated well with the increase in residual DSBs. The two cell lines, however, did not vary in cell recovery. For sublethal as well as potentially lethal damage, Rat-1 and R7080 cells showed identical recovery ratios. These data demonstrate that the overexpression of human Ku70/Ku80 led to a reduced capacity for DSB repair with an associated increase in cell sensitivity but with no effect on cell recovery.     

白念珠菌DNA损伤与修复

内外环境中各种因素如电离辐射、紫外辐射、氧化剂、烷化剂等都可以造成白念珠菌DNA的损伤。如果DNA的损伤得不到有效的修复,便会造成突变。白念珠菌的突变率很高,但并不是所有DNA受损伤的细胞都会表现出突变型性状,这跟其自身的修复系统有很大关系,主要包括切除修复、错配修复及双链断裂修复等途径,使得绝大多数损伤能够及时修复,从而维持DNA的完整性与稳定性。白念珠菌DNA的损伤修复可能影响其适应性、药物敏感性等表型,从而给临床感染患者的治疗增加难度。本文主要从白念珠菌DNA损伤的产生,损伤信号的传导识别及损伤修复三方面综述目前的研究进展。     

Interaction function gamma(x) for Chinese hamster cells treated with hypertonic phosphate-buffered saline after irradiation

The repair of potentially lethal damage (PLD) in stationary-phase V79 Chinese hamster cells, which was expressible by a postirradiation treatment with hypertonic (0.5 M NaCl) phosphate-buffered saline (PBS), was analyzed within the framework of the theory of dual radiation action. The interaction function gamma(x) was estimated for cells permitted to repair PLD for various intervals of time. The experimental data indicated that 50-60% of the lethal lesions produced at the time of irradiation were repaired in 120 min. The repair of PLD was implicitly involved in the probability of the interaction of sublesions. That is, g(x,trep) was defined as the probability that two sublesions separated by distance x interact to produce a lethal lesion which will not be repaired until the fixation by treatment with hypertonic PBS at time trep after irradiation. It is concluded that the time dependence of the repair of PLD is not independent of the interaction distance x. Three conclusions are drawn: (1) The repair of a lesion produced by a long distance interaction is not detectable by postirradiation treatment with hypertonic PBS. (2) A lesion produced by a short distance interaction is rapidly repaired in about 20 min. (3) A lesion produced by the interaction of sublesions separated by a distance of about 100 nm is repaired slowly.     

The role of DNA repair processes in the repair of cytogenetic disorders. The participation of slow to repair DNA damage in the repair of cytogenetic disorders

Caffeine was used to study the kinetics of cytogenetic damages repair in Chinese hamster fibroblasts. Its half-time (90 min) was shown to correlate with that of repair of slowly repaired DNA damages. The caffeine-induced increase in the number of irreparable DNA damages, attributed to inhibition of double-strand break repair, is in a quantitative correlation with the effect of the cytogenetic damage modification.     

Effectivity of bacterial repair systems in near UV radiation-induced damage

Inactivation of seven strains derived fromEscherichia coli B differing in their capacity to repair damage to their DNA (exc, pol, rec) after irradiation with far (254 nm) and middle and near (300 to 380 and 320–400 nm) UV light was investigated. The same bacterial strains were also used as hosts for the UV-irradiated pliage T7. The damage induced in bacteria and the phage by the near UV radiation was repaired only to a lesser extent by the investigated repair mechanisms or was not repaired at all.     

抗辐射菌中DNA损伤修复主要基因群的研究进展

抗辐射红色球菌对电离辐射具有很高的放射线抵抗性,该菌具有惊人的DNA的二条链切断的修复能力,由辐射等引起的切断损伤DNA在几至十几小时内能高效正确地进行完全修复。在对切断的双链DNA进行修复时,除了大肠杆菌等生物在切断的双链DNA修复时出现的蛋白质以外,还有该菌所特有的修复蛋白质也参与修复。本文对该菌所特有的DNA二条链的切断损伤修复的主要基因及其相互作用进行了简要介绍。     

Repair of HZE-particle-induced DNA double-strand breaks in normal human fibroblasts

DNA damage generated by high-energy and high-Z (HZE) particles is more skewed toward multiply damaged sites or clustered DNA damage than damage induced by low-linear energy transfer (LET) X and gamma rays. Clustered DNA damage includes abasic sites, base damages and single- (SSBs) and double-strand breaks (DSBs). This complex DNA damage is difficult to repair and may require coordinated recruitment of multiple DNA repair factors. As a consequence of the production of irreparable clustered lesions, a greater biological effectiveness is observed for HZE-particle radiation than for low-LET radiation. To understand how the inability of cells to rejoin DSBs contributes to the greater biological effectiveness of HZE particles, the kinetics of DSB rejoining and cell survival after exposure of normal human skin fibroblasts to a spectrum of HZE particles was examined. Using gamma-H2AX as a surrogate marker for DSB formation and rejoining, the ability of cells to rejoin DSBs was found to decrease with increasing Z; specifically, iron-ion-induced DSBs were repaired at a rate similar to those induced by silicon ions, oxygen ions and gamma radiation, but a larger fraction of iron-ion-induced damage was irreparable. Furthermore, both DNA-PKcs (DSB repair factor) and 53BP1 (DSB sensing protein) co-localized with gamma-H2AX along the track of dense ionization produced by iron and silicon ions and their focus dissolution kinetics was similar to that of gamma-H2AX. Spatial co-localization analysis showed that unlike gamma-H2AX and 53BP1, phosphorylated DNA-PKcs was localized only at very specific regions, presumably representing the sites of DSBs within the tracks. Examination of cell survival by clonogenic assay indicated that cell killing was greater for iron ions than for silicon and oxygen ions and gamma rays. Collectively, these data demonstrate that the inability of cells to rejoin DSBs within clustered DNA lesions likely contributes to the greater biological effectiveness of HZE particles.     

Comet assay evaluation of DNA single- and double-strand breaks induction and repair in C3H10T1/2 cells

Ionizing radiation is a potent inducer of DNA damage because it causes single- and double-strand breaks, alkali-labile sites, base damage, and crosslinks. The interest in ionizing radiation is due to its environmental and clinical implications. Single-strand breaks, which are the initial damage induced by a genotoxic agent, can be used as a biomarker of exposure, whereas the more biologically relevant double-strand breaks can be analyzed to quantify the extent of damage. In the present study the effects of ¹³⁷Cs γ-radiation at doses of 1, 5, and 10 Gray on DNA and subsequent repair by C3H10T1/2 cells (mouse embryo fibroblasts) were investigated. Two versions of the comet assay, a sensitive method for evaluating DNA damage, were implemented: the alkaline one to detect single-strand breaks, and the neutral one to identify double-strand breaks. The results show a good linear relation between DNA damage and radiation dose, for both single-strand and double-strand breaks. A statistically significant difference with respect to controls was found at the lowest dose of 1 Gy. Heterogeneity in DNA damage within the cell population was observed as a function of radiation dose. Repair kinetics showed that most of the damage was repaired within 2 h after irradiation, and that the highest rejoining rate occurred with the highest dose (10 Gy). Single-strand breaks were completely repaired 24 h after irradiation, whereas residual double-strand breaks were still present. This finding needs further investigation. This revised version was published online in July 2006 with corrections to the Cover Date.