Initial Rate Of Repair Research Articles

Direct visualization of repair of oxidative damage by OGG1 in the nuclei of live cells

Oxidative DNA damage caused by intracellular reactive oxygen species (ROS) is widely considered to be important in the pathology of a range of human diseases including cancer as well as in the aging process. A frequently occurring mutagenic base lesion produced by ROS is 8-oxo deoxyguanine (8-oxo dG) and the major enzyme for repair of 8-oxo dG is 8-oxoguanine-DNA glycosylase 1 (OGG1). There is now substantial evidence from bulk biochemical studies that a common human polymorphic variant of OGG1 (Ser326Cys) is repair deficient, and this has been linked to individual risk of pathologies related to oxidative stress. In the current study, we have used the technique of multiphoton microscopy to induce highly localized oxidative DNA damage in discrete regions of the nucleus of live cells. Cells transfected with GFP-tagged OGG1 proteins demonstrated rapid (<2 min) accumulation of OGG1 at sites of laser-induced damage as indicated by accumulation of GFP-fluorescence. This was followed by repair as evidenced by loss of the localized fluorescence over time. Quantification of the rate of repair confirmed that the Cys326 variant of OGG1 is repair deficient and that the initial repair rate of damage by Cys326 OGG1 was 3 to 4 fold slower than that observed for Ser326 OGG1. These values are in good agreement with kinetic data comparing the Ser326 and Cys326 proteins obtained by biochemical studies.

5,6-Dihydrothymine Impairs the Base Excision Repair Pathway of a Closely Opposed AP Site or Single-Strand Break

Ionizing radiation can induce clustered DNA damage (two or more lesions formed within one to two helical turns of DNA by passage of a single ionization track). Using oligonucleotide constructs containing clustered DNA lesions at defined positions, evidence is presented demonstrating that a persistent 5,6-dihydrothymine (DHT) lesion reduces the efficiency of rejoining, in mammalian nuclear extracts, of an opposing AP site or SSB when within 5 bp. The efficiency of repair of the SSB is reduced when DHT is present on the opposing strand in both the 3' and 5' orientation; however, the efficiency of the repair of the AP site is reduced only when DHT is present 3' to the AP site. DNA polymerase beta and ligation are particularly impaired by DHT. It was also shown that in the presence of DHT there is a greater dependence on the long-patch base excision repair pathway than when DHT is absent. In addition, immunodepletion of XRCC1 from the nuclear extracts slows down the initial rate of repair of the AP site in both the presence and absence of DHT, but immunodepletion of XRCC1 has no influence on the repair of an SSB.

Repair of radiation-induced DNA double-strand breaks is dependent upon radiation quality and the structural complexity of double-strand breaks.

Mammalian cells primarily repair DSBs by nonhomologous end joining (NHEJ). To assess the ability of human cells to mediate end joining of complex DSBs such as those produced by chemicals, oxidative events, or high- and low-LET radiation, we employed an in vitro double-strand break repair assay using plasmid DNA linearized by these various agents. We found that human HeLa cell extracts support end joining of complex DSBs and form multimeric plasmid products from substrates produced by the radiomimetic drug bleomycin, 60Co gamma rays, and the effects of 125I decay in DNA. End joining was found to be dependent on the type of DSB-damaging agent, and it decreased as the cytotoxicity of the DSB-inducing agent increased. In addition to the inhibitory effects of DSB end-group structures on repair, NHEJ was found to be strongly inhibited by lesions proximal to DSB ends. The initial repair rate for complex non-ligatable bleomycin-induced DSBs was sixfold less than that of similarly configured (blunt-ended) but less complex (ligatable) restriction enzyme-induced DSBs. Repair of DSBs produced by gamma rays was 15-fold less efficient than repair of restriction enzyme-induced DSBs. Repair of the DSBs produced by 125I was near the lower limit of detection in our assay and was at least twofold lower than that of gamma-ray-induced DSBs. In addition, DSB ends produced by 125I were shown to be blocked by 3'-nucleotide fragments: the removal of these by E. coli endonuclease IV permitted ligation.

Fidelity of uracil-initiated base excision DNA repair in DNA polymerase beta-proficient and -deficient mouse embryonic fibroblast cell extracts.

Uracil-initiated base excision DNA repair was conducted using homozygous mouse embryonic fibroblast DNA polymerase beta (+/+) and (-/-) cells to determine the error frequency and mutational specificity associated with the completed repair process. Form I DNA substrates were constructed with site-specific uracil residues at U.A, U.G, and U.T targets contained within the lacZalpha gene of M13mp2 DNA. Efficient repair was observed in both DNA polymerase beta (+/+) and (-/-) cell-free extracts. Repair was largely dependent on uracil-DNA glycosylase activity because addition of the PBS-2 uracil-DNA glycosylase inhibitor (Ugi) protein reduced ( approximately 88%) the initial rate of repair in both types of cell-free extracts. In each case, the DNA repair patch size was primarily distributed between 1 and 8 nucleotides in length with 1 nucleotide repair patch constituting approximately 20% of the repair events. Addition of p21 peptide or protein to DNA polymerase beta (+/+) cell-free extracts increased the frequency of short-patch (1 nucleotide) repair by approximately 2-fold. The base substitution reversion frequency associated with uracil-DNA repair of M13mp2op14 (U.T) DNA was determined to be 5.7-7.2 x 10(-4) when using DNA polymerase beta (+/+) and (-/-) cell-free extracts. In these two cases, the error frequency was very similar, but the mutational spectrum was noticeably different. The presence or absence of Ugi did not dramatically influence either the error rate or mutational specificity. In contrast, the combination of Ugi and p21 protein promoted an increase in the mutation frequency associated with repair of M13mp2 (U.G) DNA. Examination of the mutational spectra generated by a forward mutation assay revealed that errors in DNA repair synthesis occurred predominantly at the position of the U.G target and frequently involved a 1-base deletion or incorporation of dTMP.

The repair of photosystem II is the site of regulation by environmental stresses

The photosynthetic machinery, in particular the photosystem II complex (PSII), is rapidly damaged by light, but it is also rapidly repaired by light in vivo. Therefore, the extent of photodamage in living organisms is a result of balance between the photo-induced damage to PSII and the repair of the photodamaged PSII. We developed a method to separately measure the damage and repair processes in the cyanobacterium Synechocystis sp. PCC 6803. The initial rate of strong light-induced inactivation of PSII was measured in the presence of lincomycin which blocks the repair process by inhibiting the protein synthesis, whereas the initial rates of weak light-induced recovery of PSII activity was measured after switching the light intensity from strong to weak. We observed that the initial rate of photodamage was proportional to light intensity and this proportionality was not affected by the presence of DCMU or reactive oxygen species (ROS) such as superoxide radicals and H2O2 and by stress conditions due to high salt and cold. In contrast, the initial rate of repair was fast at low intensities of light and was saturated at 300 µE m-2 s-1, and this rate of repair at the saturation level was depressed by the environmental factors. Based on these observations we conclude that the photodamage to PSII depends solely on light intensity without regulation and that the repair of the PSII is the site of regulation by environments. The results of Northern blotting and Western blotting analyses suggest that the major site of inhibitory action by environmental factors is the translation of psbA genes.

The yeast RAD2, but not RAD1, gene is involved in the transcription-coupled repair of thymine glycols.

Transcription-coupled repair of H2O2-induced thymine glycols and UV-induced pyrimidine dimers has been shown to occur in the yeast Saccharomyces cerevisiae. In order to determine whether the preferential repair of thymine glycols is carried out by the same nucleotide excision repair complex that removes pyrimidine dimers, we examined the repair of thymine glycols in two yeast mutants, rad1 delta and rad2 delta, in which the nucleotide excision repair pathway was disrupted. We find that in both wild-type and a rad1 delta mutant, repair of thymine glycols occurs faster on the transcribed strand of the GAL7 gene than on the nontranscribed strand. This indicates that transcription-coupled repair can occur in the absence of nucleotide excision repair and that repair of oxidative DNA damage initiated by an N-glycosylase can be coupled to transcription. In contrast, the initial rate of repair of thymine glycols on the transcribed strand of the GAL7 gene in a rad2 delta mutant is significantly slower than that for the wild-type cells, with kinetics similar to that of the nontranscribed strand. However, by 60 min post-treatment, the amount of repair on the transcribed strand in the rad2 delta eventually reaches that of the wild-type cells. We conclude that repair of oxidative DNA damage, such as thymine glycols, can be coupled to transcription and that RAD2 facilitates transcription-coupled repair of oxidative DNA damage in yeast.

Induction and Repair of DNA Double-Strand Breaks in the Same Dose Range as the Shoulder of the Survival Curve

We have used pulsed-field gel electrophoresis (PFGE) to test two hypotheses that have been proposed to explain the survival curves with shoulders which are characteristic of low-LET ionizing radiation: (1) Neutral elution studies of the induction of double-strand breaks (DSBs) have suggested that ionizing radiation might induce DSBs in a nonlinear fashion at low doses. (2) Based on analogies to enzyme kinetics, DSB repair might be saturating in the shoulder region. We quantified DSB induction and survival resulting from doses between 0 and 5 Gy spanning the shoulder region of the survival curve. We found that DSB induction was linear at all doses tested down to 0.5 Gy, the limits of sensitivity. Therefore, nonlinear DSB induction cannot account for the shape of the survival curve. To determine whether the DSB repair system was saturated in the shoulder region, we quantified the rate of DSB repair as a function of dose of X rays between 1.25 and 20 Gy. The repair of DSBs was exponential with half-times of repair constant for doses below 10 Gy, and averaged 28 min. We determined the initial rate of repair from the exponential repair kinetics for each dose. The initial rate of repair after radiation treatment increased linearly with dose up to at least 10 Gy. Therefore, saturating DSB repair cannot explain the shoulder of the survival curve.

Induction and repair of DNA single-strand breaks and DNA base damage at different cellular stages of spermatogenesis of the hamster upon in vitro exposure to ionizing radiation

Alkaline elution has been used for quantitative detection of DNA damage caused by ionizing radiation in unlabeled somatic and germ cells. Both the induction and subsequent repair have been studied for two classes of DNA damage, viz. single-strand breaks (SSB), and base damage (BD) recognized by the γ-endonuclease activity in a cell-free extract of Micrococcus luteus bacteria. The high sensitivity of the assay permitted the measurement of induction and repair of SSB and BD after in vitro exposure of hamster germ cells in different cellular stages of spermatogenesis (spermatocytes, round and elongated spermatids), and of bone-marrow cells, to biologically relevant doses (0–8 Gy) of 60Co γ-rays. A dose-dependent increase was observed for both types of lesions, which was similar for most cell types. The elongated spermatids, however, showed a lower induction frequency of SSB (and perhaps BD).Spermatocytes, round spermatids and bone-marrow cells had normal, fast repair of the SSB when compared with the repair reported for cultured rodent cells and human lymphocytes. In contrast, the elongated spermatids showed hardly any SSB repair. The initial rate of repair of BD in spermatocytes and bone-marrow cells was in the same range as that for SSB, but only 60–70% of the initial BD was repaired within 1 h, whereas after that period no SSB were detectable. The round spermatids hardly repaired any BD within the first hour after irradiation, but after 7 h only a few BD could be detected. In elongated spermatids repair of BD could not be measured due to a high background level of this type of damage.

Repair of a synthetic abasic site involves concerted reactions of DNA synthesis followed by excision and ligation.

A synthetic analog of an abasic site in DNA is efficiently repaired by a short-patch repair mechanism in soluble extracts of Xenopus laevis oocytes (Y. Matsumoto and D. F. Bogenhagen, Mol. Cell. Biol. 9:3750-3757, 1989). We present a detailed analysis of the repair mechanism, using extracts depleted of endogenous nucleotide pools. ATP was required for repair with a sharp optimal concentration of 5 mM. The initial rate of repair was increased by preincubation of the DNA in the extract in the presence of ATP. During this preincubation, the DNA was cleaved on the 5' side of the lesion by a class II apurinic-apyrimidinic endonuclease, but removal of the abasic sugar residue was not observed prior to addition of deoxynucleotides to the reaction. Immediately following DNA synthesis, excision and ligation proceeded in a coordinated manner to complete repair. DNA preincubated in the extract in the absence of deoxynucleotides remained associated with repair enzymes during gel filtration. These observations suggest that the enzymes involved in concerted repair of the abasic site form a complex on DNA.

Repair of a Synthetic Abasic Site Involves Concerted Reactions of DNA Synthesis Followed by Excision and Ligation

A synthetic analog of an abasic site in DNA is efficiently repaired by a short-patch repair mechanism in soluble extracts of Xenopus laevis oocytes (Y. Matsumoto and D. F. Bogenhagen, Mol. Cell. Biol. 9:3750-3757, 1989). We present a detailed analysis of the repair mechanism, using extracts depleted of endogenous nucleotide pools. ATP was required for repair with a sharp optimal concentration of 5 mM. The initial rate of repair was increased by preincubation of the DNA in the extract in the presence of ATP. During this preincubation, the DNA was cleaved on the 5' side of the lesion by a class II apurinic-apyrimidinic endonuclease, but removal of the abasic sugar residue was not observed prior to addition of deoxynucleotides to the reaction. Immediately following DNA synthesis, excision and ligation proceeded in a coordinated manner to complete repair. DNA preincubated in the extract in the absence of deoxynucleotides remained associated with repair enzymes during gel filtration. These observations suggest that the enzymes involved in concerted repair of the abasic site form a complex on DNA.

Differential repair of DNA damage in the human metallothionein gene family.

We studied the repair of UV- and aflatoxin B1 (AFB1)-induced damage in the human metallothionein (hMT) gene family. After exposure to either UV or AFB1, DNA damage was initially repaired faster in the DNA fragments containing the transcribed hMT-IA, hMT-IE, and hMT-IIA genes than in the genome overall. By 6 h posttreatment, there was at least twice as much repair in these genes as in the rest of the genome. Repair of UV damage in the hMT-IB gene, which shows cell-type specific expression, and in the hMT-IIB gene, which is a nontranscribed processed pseudogene, was about the same as in the rest of the genome, whereas repair of AFB1-induced damage was deficient in these two genes. Inducing transcription of the three expressed hMT genes with CdCl2 or of only the hMT-IIA gene with dexamethasone increased the initial rate of repair in the induced genes another twofold over the rate observed when they were transcribed at a basal level. The rates of repair in the hMT-IB and hMT-IIB genes were not altered by these inducing treatments. Transcription of the hMT genes was transiently inhibited after UV irradiation. Inducing transcription of the genes did not shorten this UV-induced delay. Thus, the efficiency of repair of damage in a DNA sequence is dependent on the level of transcriptional activity associated with that sequence. However, an increased efficiency in repair of a gene itself is not necessarily coupled to recovery of its transcription after DNA damage.

Differential repair of DNA damage in the human metallothionein gene family.

We studied the repair of UV- and aflatoxin B1 (AFB1)-induced damage in the human metallothionein (hMT) gene family. After exposure to either UV or AFB1, DNA damage was initially repaired faster in the DNA fragments containing the transcribed hMT-IA, hMT-IE, and hMT-IIA genes than in the genome overall. By 6 h posttreatment, there was at least twice as much repair in these genes as in the rest of the genome. Repair of UV damage in the hMT-IB gene, which shows cell-type specific expression, and in the hMT-IIB gene, which is a nontranscribed processed pseudogene, was about the same as in the rest of the genome, whereas repair of AFB1-induced damage was deficient in these two genes. Inducing transcription of the three expressed hMT genes with CdCl2 or of only the hMT-IIA gene with dexamethasone increased the initial rate of repair in the induced genes another twofold over the rate observed when they were transcribed at a basal level. The rates of repair in the hMT-IB and hMT-IIB genes were not altered by these inducing treatments. Transcription of the hMT genes was transiently inhibited after UV irradiation. Inducing transcription of the genes did not shorten this UV-induced delay. Thus, the efficiency of repair of damage in a DNA sequence is dependent on the level of transcriptional activity associated with that sequence. However, an increased efficiency in repair of a gene itself is not necessarily coupled to recovery of its transcription after DNA damage.

Radiosensitization, Thiol Oxidation, and Inhibition of DNA Repair by SR 4077

The mechanism of radiosensitization by diazenedicarboxylic acid bis(N),N-piperidide (SR 4077), a less toxic analog of diamide, was studied using Chinese hamster ovary cells. SR 4077 gave an average SER of 1.58 for postirradiation incubations of 0.5, 1.0, or 2.0 h. Intracellular GSH and protein thiols decreased rapidly following drug addition and GSSG increased. The GSH/GSSG ratio shifted to 1/1.6 after SR 4077 addition but returned to greater than 10/1 between 0.5 and 1.0 h. After 4 h, total intracellular GSH was only 58% of pretreatment level and extracellular GSSG increased. Protein thiols decreased to 18% of pretreatment values, recovered most rapidly between 0.5 and 1.0 h, and reached 87% of pretreatment level after 4 h. A decrease in DNA single-strand break repair as measured by alkaline filter elution rate over 0.5 h was seen, and the initial rate of repair was slower than in cells not treated with SR 4077. DNA double-strand break repair as measured by neutral filter elution rate was delayed during the first hour after irradiation when cells were treated with SR 4077. The times for maximum radiosensitization, GSH and protein thiol oxidation and recovery, and DNA strand break repair kinetics were closely linked. We propose that a protein thiol(s) required in repair processes was reversibly oxidized during SR 4077 treatment.

Measurement of the kinetics of DNA repair synthesis after uv irradiation using immunochemical staining of incorporated 5-bromo-2′-deoxyuridine and flow cytometry

The kinetics of unscheduled DNA synthesis in normal human fibroblasts was characterized by flow cytometry utilizing the immunofluorescent detection of 5-bromo-2′-deoxyuridine (BrdUrd) incorporated into cellular DNA during the repair process. Quiescent normal human fibroblasts were irradiated with ultraviolet light and incubated in the presence of BrdUrd during a postirradiation repair period. The amount of unscheduled DNA synthesis was then quantified in the quiescent cells by immunofluorescence staining using monoclonal antibodies against BrdUrd incorporated into the DNA. Significant amounts of unscheduled DNA synthesis were measured after doses as low as 0.1 J/m 2 and for time periods as short as 15 min. The initial repair rate was found to be linear with time at all doses tested until repair neared completion. Interestingly, the initial repair rate was constant for doses over the range of 5 to 40 J/m 2, whereas the time to completion of repair was dose dependent. These results suggest that above 5 J/m 2 in normal human fibroblasts, the repair process is saturated but continues to function until all available regions are repaired. Using this methodology for measuring unscheduled DNA synthesis in combination with second and third flow markers, it is now possible to measure unscheduled DNA synthesis in heterogeneous mixtures of cells.

A Calmodulin Antagonist Has No Effect on the Repair of X-Ray-Induced Damage in a Murine Mammary Carcinoma Cell Line

The effects of the calmodulin antagonist W13 were determined on potentially lethal damage repair, sublethal damage repair, and X-ray-induced DNA damage repair following X irradiation of 67 murine mammary carcinoma cells in the proliferative and quiescent states. Studies with W13 (20 micrograms/ml) on proliferating cells showed that the cells rounded up within 2 h but stayed attached to the dishes and there was a slight transient G2 block by 6 h. Also, the proportion of S-phase cells at 12 h was reduced to 65% of control with the concurrent [3H]thymidine incorporation reduced to 62% of control. There was no detectable effect from this pharmacological dose of W13 either on PLDR in proliferating cells at 400 and 800 rad or on quiescent cells at 200 and 400 rad. Likewise, there was no measurable effect on SLDR in either proliferating or quiescent cells at equally split doses of 800 and 600 rad, respectively. In addition, for control vs W13-treated proliferating cells, no difference was detected either in the induction of DNA damage by X irradiation or in the initial rate of repair (T 1/2 approximately equal to 7 min), as measured by the alkaline filter elution assay. In contrast to uv and bleomycin-induced damage, these data suggest that calmodulin may have no major role in either the molecular or cellular recovery from X-ray-induced damage in mammalian cells.

The rate of removal of pyrimidine dimers in quiescent cultures of normal human and xeroderma pigmentosum cells

The rate of removal of pyrimidine dimers from DNA of UV (254 nm)-irradiated (1 J/m 2) normal and xeroderma pigmentosum (XP) cells maintained in culture as nondividing populations was determined. Several normal and XP strains from complementation groups A, C and D were studied. The excision rates and survival ability of nondividing cells were examined to determine if an abnormal sensitivity was associated with a decreased rate of dimer excision. The results show that all normal strains studied excise pyrimidine dimers at the same rate, with the rate curve characterized by two components. All ‘excision-deficient’ XP strains excise dimers at a slower-than-normal rate, with the rate curves also characterized by two components. The rate constants for the first components of all of the XP strains (group A, C and D) are the same, one tenth of the normal rate constant, except for XP8LO (group A). XP8LO has a first-component rate constant similar to that of normal strains and a second component rate constant similar to that of other group A strains (XP12BE, XP25RO). Thus, the slower rate of dimer excision in XP8LO is due to a defect in the mechanism responsible for the second component of the excision-rate curve. In general, an abnormal sensitivity of nondividing cells to UV is associated with a reduced dimer-excision rate. A notable exception to this is the group C strain XP1BE which has an initial repair rate similar to that of group A XP12BE but is considerably more resistant when survival is measured.

Hyperthermic Potentiation of the Effects of a Clinically Significant X-Ray Dose on Cell Survival, DNA Damage, and DNA Repair

Mouse leukemia L1210 cells grown in suspension were used to study the synergistic effects of combined hyperthermia (43, 45.5°C) followed immediately by clinically significant X-ray doses (3-6 Gy). Cell survival was determined by growth in soft agar, and the extent and repair of DNA single-strand breaks were measured by the alkaline elution technique. The degree of synergism for cytotoxic effects of heat and radiation (3 Gy) increased as a function of treatment time at 43 or 45.5°C. Hyperthermia showed a greater synergistic effect with X rays (3-6 Gy) at 43°C than at 45.5°C when compared at equal heat reductions in survival. The initial rate of repair of DNA single-strand breaks was inhibited and the extent of repair failed to reach 100% by 4 hr with combined hyperthermia and the 3-Gy X-ray dose. These combined treatments showed synergism in terms of reducing cell survival. The reason for the incomplete repair of X-ray-induced DNA strand breaks with prior hyperthermic treatment was that the hyperthermia it...

Increase of UV-induced DNA repair synthesis during blast transformation of human lymphocytes

UV-induced DNA repair synthesis, measured as unscheduled DNA synthesis, was studied in human peripheral lymphocytes in various phases of the cell cycle. Mitogen transformation of the lymphocytes was effected with phytohemagglutinin (PHA), and the stage in the cell cycle was determined by measuring the Feulgen DNA content and the dry mass in individual cells by cytophotometry. The initial rate of repair was determined by autoradiography after UV-light irradiation (19.2 J/m 2) and incubation of the cells for 30 min with [ 3H]thymidine. When the cells progressed from the G0 to the G1 phase there was a 3-fold increase in the grain count. The correlation between the grain count and the dry mass indicated an increase in the initial rate of repair during the progression of cells from G0 to G2 phase. G2 cells were more heavily labelled than those in G1, but there did not seem to be any difference between these two phases as regards the relationship between grain count and DNA content. The results indicate that the initial rate of UV-induced DNA repair may differ in various phases of the lymphocyte cell cycle.