X-irradiation sensitivity in Escherichia coli defective in DNA replication

Summary A mutant of Escherichia coli with a temperature sensitive defect in DNA replication is sensitive to X-irradiation but not to UV-irradiation. After UV-irradiation, dark-repair processes—dimer excision, DNA breakdown, repair synthesis and DNA strand joining—appear normal at the restrictive temperature. After X-irradiation, DNA degradation exceeds that in the wild type, and irradiation-dependent DNA synthesis does not occur. Single-strand breaks introduced into the DNA by the irradiation are nor repaired. The data indicate that the mutation results in a defect in repair of X-ray induced single-strand breaks as well as a defect in DNA replication. They provide evidence for the existence of a repair pathway for X-irradiated DNA similar to, but at least partially independent from, that postulated for the dark-repair of UV-irradiated DNA, viz., degradation at the site of the lesion, resynthesis of the degraded DNA complement and ligation of the DNA strand.This material has been published as an abstract in Genetics 64, p. 18 (1970).     

ATP-type DNA ligase requires other proteins for its activity in vitro and its operon components for radiation resistance in Deinococcus radiodurans in vivo

A multiprotein DNA processing complex isolated from Deinococcus radiodurans contains the DNA repair protein PprA, an ATP-type DNA repair ligase (LigB) encoded by the drB0100 gene, and protein kinase activity. An ATP-dependent DNA end-joining activity was detected in the complex. To elucidate the function of the drB0100 gene, we generated the deletion mutant for the DR_B0100 ORF. The mutant exhibited a nearly 2-log cycle reduction in growth rate when exposed to a 10,000 Gray dose of γ-radiation, and a significant loss in mitomycin C and methylmethane sulphonate tolerance as compared with wild type. Functional complementation of these phenotypes required the wild-type copy of drB0100 along with other genes such as drb0099 and drb0098, organized downstream in the operon. The in vitro DNA ligase activity of LigB was stimulated severalfold by PprA in the presence of the recombinant DRB0098 protein. However, this activity did not improve when PprA was substituted with purified DRB0099 protein or when DRB0098 protein was substituted with the DRB0099 protein in the presence of PprA in solution. These results suggest that PprA and DRB0098 protein are required for LigB function. Furthermore, they also suggest that the LigB operon components contribute to radiation resistance and double-strand break (DSB) repair in D. radiodurans.     

Induction of DNA double-strand breaks by X-rays in a radiosensitive strain of the yeast Saccharomyces cerevisiae

Sedimentation profiles for chromosomal DNA from unirradiated and X-irradiated yeast cells of wild type and rad 52 strains are presented. These profiles indicate that, whereas wild type strains rejoin DNA double-strand breaks, rad 52 strains apparently do not. These data suggest that the rad 52 mutant lacks a repair system for X-ray induced damage and are consistent with the proposal that an unrepaired chromosome break leads to reproductive cell death.     

Genetic control of the processes of postradiation repair of a compact chromosome in Micrococcus radiodurans cells

X-irradiation of Micrococcus radiodurans cells with sublethal doses caused disturbances in the structure of a membrane-bound compact chromosome. Recovery of the compact chromosome occurred during the postirradiation incubation of the wild type cells and cells of the UVS-17 mutant deficient in DNA-polymerase. This process was blocked in cells of rec-30 mutant with the impaired system of genetic recombination: this is indicative of an important role played by rec-30 gene product in the postirradiation recovery of the compact chromosome in M. radiodurans cells.     

Role for deoxyribonucleic acid ligase in deoxyribonucleic acid polymerase i-dependent repair synthesis in toluene-treated escherichia coli.

In a toluene-treated mutant of Escherichia coli K-12 having a temperature-sensitive, conditionally lethal mutation in the structural gene for deoxyribonucleic acid (DNA) ligase, an extensive DNA repair synthesis occurred in X-irradiated cells at the nonpermissive temperature, 42 C. At the permissive temperature, 30 C, nearly normal semiconservative synthesis and limited repair synthesis were observed when DNA ligase was activated by the addition of nicotinamide adenine dinucleotide.     

DNA polymerase I-mediated ultraviolet repair synthesis in toluene-treated Escherichia coli.

DNA synthesis after ultraviolet irradiation is low in wild type toluene-treated cells. The level of repair incorporation is greater in strains deficient in DNA polymerase I. The low level of repair synthesis is attributable to the concerted action of DNA polymerase I and polynucleotide ligase. Repair synthesis is stimulated by blocking ligase activity with the addition of nicotinamide mononucleotide (NMN) or the use of a ligase temperature-sensitive mutant. NMN stimulation is specific for DNA polymerase I-mediated repair synthesis, as it is absent in isogenic strains deficient in the polymerase function or the 5' leads to 3' exonuclease function associated with DNA polymerase I. DNA synthesis that is stimulated by NMN is proportional to the ultraviolet exposure at low doses, nonconservative in nature, and is dependent on the uvrA gene product but is independent of the recA gene product. These criteria place this synthesis in the excision repair pathway. The NMN-stimulated repair synthesis requires ATP and is N-ethylmaleimide-resistant. The use of NMN provides a direct means for evaluating the involvement of DNA polymerase I in excision repair.     

Effect of cordycepin(3'-deoxyadenosine) on excision repair of 5,6-dihydroxy-dihydrothymine-type products from the DNA of Micrococcus radiodurans

Cordycepin(3'-deoxyadenosine), a nucleoside analog, has been shown to enhance radiation-induced cell killing. In an effort to elucidate the possible mechanism for enhancement of cell killing, the effect of cordycepin on the excision repair of radiation-induced 5,6-dihydroxy-dihydrothymine-type (t') products from the DNA of wild type Micrococcus radiodurans was investigated. The capacity of M. radiodurans to excise nondimeric (t') products from its DNA was significantly impaired after cordycepin treatment. The results suggest that the increased radiation sensitivity of cordycepin-treated cells could be due to alterations in cellular processes that repair DNA damage.     

Repair and recombination of X-irradiated plasmids in Xenopus laevis oocytes.

Plasmid DNA substrates were X-irradiated and injected into the nuclei of Xenopus laevis oocytes. After incubation for 20 h, DNA was recovered from the oocytes and analyzed simultaneously for repair and for intermolecular homologous recombination by electrophoresis and bacterial transformation. Oocyte-mediated repair of DNA strand breaks was observed with both methods. Using a repair-deficient mutant Escherichia coli strain and its repair-proficient parent as hosts for the transformation assay, we also demonstrated that oocytes repaired oxidative-type DNA base damage induced by X-rays. X-irradiation of a circular DNA stimulated its potential to recombine with a homologous linear partner. Recombination products were detected directly by Southern blot hybridization and as bacterial transformant clones expressing two antibiotic resistance markers originally carried separately on the two substrates. The increase in recombination was dependent on X-ray dose. There is some suggestion that lesions other than double-strand breaks contribute to the stimulation of oocyte-mediated homologous recombination. In summary, oocytes have considerable capacity to repair X-ray-induced damage, and some X-ray lesions stimulate homologous recombination in these cells.     

Identification of a DNA processing complex from Deinococcus radiodurans

An efficient DNA strand break repair contributes to the radioresistance of Deinococcus radiodurans, which harbors the DNA repair pathways nearly identical to Escherichia coli. The molecular mechanisms of these proteins functioning in 2 diverse classes of bacteria seem to be different. The macromolecular interactions and formation of multiprotein complexes in vivo have gained significant importance in explaining the mechanism of the complex cellular processes. Here, we report the identification of a novel DNA metabolic protein complex from D. radiodurans. A similar complex has, however, not been found in E. coli. Mass spectrometric analysis showed the presence of a few known DNA repair proteins, molecular chaperones, and a large number of uncharacterized proteins from D. radiodurans R1. Biochemical and immunoblotting results indicated the presence of the protein promoting DNA repair A, DNA polymerase, Mg2+, and (or) Mn2+ -dependent 5'-->3' exonuclease activity along with protein kinase activity and phosphoproteins. DNA ligase activity was completely dependent upon the ATP requirement, as no ligase activity was seen in the presence of NAD as a cofactor. These results suggest the molecular interactions of the known DNA repair proteins with uncharacterized proteins in the macromolecular complex and the regulation of DNA degradation with the involvement of ATP and protein kinase functions.     

Properties of the nonlethal recombinational repair x and y mutants of bacteriophage T4. II. DNA synthesis.

The bacteriophage T4 recombination-deficient mutants x and y exhibited decreased rates of DNA synthesis as compared to wild-type T4. Mutant-induced DNA synthesis was more sensitive to mitomycin C than was wild-type synthesis. However, DNA synthesis in mutant- and wild-type-infected cells exhibited the same sensitivity to UV light and X-irradiation. When high-specific-activity label was administered at various times postinfection, mutant DNA synthesis resembled that of wild type for 12 min. after which time mutant-induced incorporation was greatly decreased and sensitive to mitomycin C as compared to that of the wild type. Rifampin and chloramphenicol studies indicated that the gene products necessary for synthesis measured at 15 min postinfection, including those of x+ and y+ were transcribed within 2 min and translated within 8 min postinfection. Administration of chloramphenicol to mutant x- or mutant y-infected cells exactly 8 min postinfection, however, allowed for increased synthesis at 15 min that was sensitive to mitomycin C. Cells coinfected with T4+ and T4x or T4x and T4y retained a reduced mutant-type synthesis, whereas cells coinfected with T4+ and T4y exhibited a synthesis more closely resembling that of wild type.     

Depression by NAD of x-ray-induced repair-type DNA synthesis in toluene-treated Bacillus subtilis.

NAD prevents a DNA repair-type synthesis that is dependent on polymerase I in toluene-treated, X-irradiated Bacillus subtilis. In unirradiated preparations, NAD had little effect on an ATP-dependent, semiconservative synthesis but partially inhibited a repair-type synthesis. In a mutant lacking polymerase I (polA1-), the presence of NAD did not affect dTTP utilization in DNA synthesis. Nicotinamide mononucleotide (NMN) partially reverses the NAD inhibition of repair-type DNA synthesis. NADP and FAD were ineffective as substitutes for NAD. Since NAD is the cofactor for polynucleotide ligase in Bacillus subtilis and NMN is known to discharge AMP from the active AMP ligase complex, it is proposed that activation of DNA ligase reduces dTMP incorporation by reducing sites for, or limiting DNA polymerase I action.     

Potential role for 53BP1 in DNA end-joining repair through direct interaction with DNA

Upon DNA damage, p53-binding protein 1 (53BP1) relocalizes to sites of DNA double-strand breaks and forms discrete nuclear foci, suggesting its role in DNA damage responses. We show that 53BP1 changed its localization from the detergent soluble to insoluble fraction after treatment of cells with x-ray, but not with ultraviolet or hydroxyurea. Either DNase or phosphatase treatment of the insoluble fraction released 53BP1 into the soluble fraction, showing that 53BP1 binds to chromatin in a phosphorylation-dependent manner after X-irradiation of cells. 53BP1 was retained at discrete nuclear foci in X-irradiated cells even after detergent extraction of cells, showing that the chromatin binding of 53BP1 occurs at sites of DNA double-strand breaks. The minimal domain for focus formation was identified by immunofluorescence staining of cells ectopically expressed with 53BP1 deletion mutants. This domain consisted of conserved Tudor and Myb motifs. The Tudor plus Myb domain possessed chromatin binding activity in vivo and bound directly to both double-stranded and single-stranded DNA in vitro. This domain also stimulated end-joining by DNA ligase IV/Xrcc4, but not by T4 DNA ligase in vitro. We conclude that 53BP1 has the potential to participate directly in the repair of DNA double-strand breaks.     

DNA polymerase beta promotes recruitment of DNA ligase III alpha-XRCC1 to sites of base excision repair

Base excision repair is a major pathway for the removal of simple lesions in DNA including base damage and base loss (abasic site). Base excision repair requires the coordinated action of several repair and ancillary proteins, the impairment of which can lead to genetic instability. Using a protein-DNA cross-linking assay during repair in human whole cell extracts, we monitored proteins involved in the initial steps of repair of a substrate containing a site-specific abasic site to address the molecular events following incision of the abasic site by AP endonuclease. We find that after dissociation of AP endonuclease from the incised abasic site, both DNA polymerase beta (Pol beta) and the DNA ligase IIIalpha-XRCC1 heterodimer efficiently bind/cross-link to the substrate DNA. We also find that the cross-linking efficacy of the DNA ligase IIIalpha-XRCC1 heterodimer was decreased about 2-fold in the Pol beta-deficient cell extract but was rescued by addition of purified wild type but not a mutant Pol beta protein that does not interact with the DNA ligase IIIalpha-XRCC1 heterodimer. We further demonstrate that Pol beta and the DNA ligase IIIalpha-XRCC1 heterodimer are present at equimolar concentrations in whole cell extracts and that Pol beta has a 7-fold higher affinity to the incised abasic site containing substrate than DNA ligase IIIalpha. Using gel filtration of whole cell extracts prepared at physiological salt conditions (0.15 M NaCl), we find no evidence for a stable preexisting complex of DNA Pol beta with the DNA ligase IIIalpha-XRCC1 heterodimer. Taken together, these data suggest that following incision by AP endonuclease, DNA Pol beta recognizes and binds to the incised abasic site and promotes recruitment of the DNA ligase IIIalpha-XRCC1 heterodimer through its interaction with XRCC1.     

Growth-medium-dependent repair of DNA single-strand and double-strand breaks in X-irradiated Escherichia coli

The X-ray resistance of logarithmic phase cells of Escherichia coli K-12 is enhanced threefold by growth in rich medium versus minimal medium (N. J. Sargentini, W. P. Diver, and K. C. Smith, Radiat. Res. 93, 364-380, 1983). In this work, X-ray-induced DNA strand breaks were assayed by sedimentation in alkaline and neutral sucrose gradients to correlate the enhanced survival of rich-medium-grown cells with an enhanced capacity for DNA repair. While rich-medium-grown cells showed no enhanced capacity for repairing DNA single-strand breaks in buffer, i.e., fast, polA-dependent repair, they did show an enhanced capacity to repair both single-strand and double-strand breaks in growth medium, i.e., slow, recA-dependent repair. This enhanced capacity for DNA repair in rich-medium-grown cells was inhibited by rifampicin post-treatment, indicating the requirement for de novo RNA synthesis. Kinetic studies indicated that the repair of DNA double-strand breaks was a complex process. Relative to the sedimentation rate in neutral sucrose gradients of nonirradiated DNA, the sedimentation rate of X-irradiated DNA first changed from slow to very fast. Based on alkaline sucrose gradient sedimentation studies, all the strand breaks had been repaired during the formation of the very fast sedimenting DNA. With continued incubation, the sedimentation rate of the DNA on neutral sucrose gradients decreased to the normal rate.     

Involvement of a protein kinase activity inducer in DNA double strand break repair and radioresistance of Deinococcus radiodurans

Transgenic bacteria producing pyrroloquinoline quinone, a known cofactor for dehydrogenases and an inducer of a periplasmic protein kinase activity, show resistance to both oxidative stress and protection from nonoxidative effects of radiation and DNA-damaging agents. Deinococcus radiodurans R1 encodes an active pyrroloquinoline quinone synthase, and constitutive synthesis of pyrroloquinoline quinone occurred in wild-type bacteria. Disruption of a genomic copy of pqqE resulted in cells that lacked this cofactor. The mutant showed a nearly 3-log decrease in gamma radiation resistance and a 2-log decrease in mitomycin C tolerance compared to wild-type cells. The mutant cells did not show sensitivity to UVC radiation. Expression of pyrroloquinoline quinone synthase in trans showed that there was functional complementation of gamma resistance and mitomycin C tolerance in the pqqE mutant. The sensitivity to gamma radiation was due to impairment or slow kinetics of DNA double strand break repair. Low levels of (32)P incorporation were observed in total soluble proteins of mutant cells compared to the wild type. The results suggest that pyrroloquinoline quinone has a regulatory role as a cofactor for dehydrogenases and an inducer of selected protein kinase activity in radiation resistance and DNA strand break repair in a radioresistant bacterium.     

Changes in nucleoid viscosity following X-irradiation of rat thymic and splenic cells in vitro

Summary Unscheduled DNA synthesis (UDS) suggested a higher DNA repair capacity of X-irradiated rat thymic (T) cells when compared to splenic (S) cells (Tempel 1980). In the present investigations, damage and repair of DNA supercoiling was measured in T- and S-cells following X-irradiation in vitro by using the nucleoid sedimentation technique and a simplified low-shearing viscometric test. - X-irradiation resulted in a dose (0.6–19.2 Gy) - dependent reduction in sedimentation and viscosity of nucleoids. Within a post-irradiation period of 30–45 min after a challenge dose of 19.2 Gy, DNA repair was accompanied by an increase in nucleoid sedimentation and viscosity in T-cells by about 60 and 300, in S-cells by almost 40 and 100%, resp. The increase in nucleoid viscosity within a 30 min repair period could be reduced in a concentration-dependent manner by DNA polymerase - inhibitors and proteinase K. - The higher DNA repair capacity of T-cells as reflected by UDS is confirmed therefore by the nucleoid characteristics. Apart from this suggestion, measuring nucleoid viscosity may be considered as a sensitive, simple and rapid device to detect radiation-induced DNA supercoiling phenomena.     

RecBC enzyme overproduction affects UV and gamma radiation survival of Deinococcus radiodurans

Deinococcus radiodurans recovering from the effect of acute dose of gamma (gamma) radiation shows a biphasic mechanism of DNA double strands breaks repair that involves an efficient homologous recombination. However, it shows higher sensitivity to near-UV (NUV) than Escherichia coli and lacks RecBC, a DNA strand break (DSB) repair enzyme in some bacteria. Recombinant Deinococcus expressing the recBC genes of E. coli showed nearly three-fold improvements in near-UV tolerance and nearly 2 log cycle reductions in wild type gamma radiation resistance. RecBC over expression effect on radiation response of D. radiodurans was independent of indigenous RecD. Loss of gamma radiation tolerance was attributed to the enhanced rate of in vivo degradation of radiation damaged DNA and delayed kinetics of DSB repair during post-irradiation recovery. RecBC expressing cells of Deinococcus showed wild type response to Far-UV. These results suggest that the overproduction of RecBC competes with the indigenous mechanism of gamma radiation damaged DNA repair while it supports near-UV tolerance in D. radiodurans.     

耐辐射球菌基因DRB0099缺失突变株的构建及逆境分析

耐辐射球菌(Deinococcus radiodurans R1)有着极强的辐射抗性.研究其抗辐射的机理对于处理放射性废料有着潜在的应用价值.在耐辐射球菌的基因组中,许多序列的功能未知.其中DRB0099尤为引人注意.将DRB0099缺失突变构建该基因的突变株.对野生型和突变体进行比较后发现,在正常生长条件下的前期阶段(0～16 h),突变体生长速度比野生型慢.16 h以后,野生型逐渐进入稳定生长期.这时,突变株的生长速度高于野生型.但是,野生型的浓度一直高于突变株.表明在DRB0099被删除后,耐辐射球菌的生长可能受到了阻滞.在紫外线照射的条件下,尽管野生型随着照射剂量的增加,存活率越来越低,但是要比突变体高许多.野生型具有比突变体更强的修复DNA双链断裂的能力.DRB0099可能直接参与了对DNA的修复.突变体对H2O2的敏感程度高于野生型,表明野生型耐辐射球菌在对抗活性氧保护其蛋白质、DNA或者DNA修复方面具有比突变体更强的功能.在低浓度H2O2处理条件下,尽管野生型和突变体的存活率都出现下降趋势,但二者的差值并不大.随着H2O2剂量的增加,二者的差值越来越大.表明随着活性氧浓度的增加,蛋白质和DNA损伤的数量增加,失去DRB0099基因功能的突变体比野生型更容易受到损伤.在紫外线照射处理或者H2O2处理条件下,DRB0099能够保护蛋白质和DNA.     

耐辐射球菌基因DR1709与DR2523的突变分析

摘要：【目的】检测在耐辐射球菌抵抗外来辐射和氧自由基的过程中,锰离子转运蛋白基因（DR1709和DR2523）是否发挥了作用。探讨锰离子、锰离子转运蛋白基因与耐辐射球菌辐射抗性之间的关系。【方法】分别构建这两个基因的突变体。对突变体和野生型进行紫外线照射和过氧化氢处理。对处理后的菌株存活率进行分析。【结果】DR2523被突变以后,耐辐射球菌在tryptone-glucose-yeast extract (TGY)培养液中的生长受影响很小。而DR1709突变体M1709在对数生长阶段的生长速度远低于野生型。     

The role of short-lived DNA lesions in the production of chromosome-exchange aberrations

Human lymphocytes were treated with combined UVC radiation and X-rays or they were X-irradiated and incubated for 60–90 min in the presence of DNA-repair inhibitor ara-C. The X-ray induced chromosome exchange aberration yield was enhanced both by UVC and ara-C by approximately a factor of two in the linear (low dose) portion of the dose-response curve. The enhancement was small in the dose squared (high dose) portion where previous dose-fractionation experiments have shown that X-ray-induced lesions leading to aberrations exist for several hours. The yield of aberrations in lymphocytes incubated after irradiation in the presence of ara-C reaches a saturation level almost immediately after irradiation (5–15 min). These cytogenetic observations together with a previous finding (Holmberg and Strausmanis, 1983) give direct and indirect evidence that the enhanced aberration yield is due to short-lived DNA breaks formed immediately after X-irradiation.

Measurements on the repair kinetics of the DNA breaks induced by X-irradiation show that ara-C strongly impairs the repair of short-lived X-ray-induced DNA breaks. It was also observed that the DNA breaks generated after UVC irradiation occur almost immediately after irradiation and the level of these transient DNA breaks reaches saturation even for short incubation times. Thus, the repair of these breaks can compete with the repair of short-lived X-ray-induced DNA-breaks in combined irradiation with UVC and X-rays.

The experimental results can be explained on the assumption that X-ray-induced aberrations originate from exchange complexes formed in interactions between both short-lived DNA breaks. The short-lived DNA breaks give rise to exchange complexes mainly within single ionization tracks where the DNA breaks are close together. The time between irradiation and exchange complex formation is of the order of 5–15 min within such a track, and short-lived breaks might be repaired before complexes have been formed. If the DNA repair of these breaks is delayed by UVC or ara-C treatment this results in a higher probability of exchange-complex formation. In contrast, interactions between breaks in different tracks originate from long-lived DNA breaks and the probability for complex formation from these breaks is not markedly affected by UVC or ara-C.