Effect of Caffeine on Postreplication Repair in Human Cells

DNA synthesized shortly after ultraviolet (UV) irradiation of human cells is made in segments that are smaller than normal, but at long times after irradiation the segments made are normal in size. Upon incubation, both the shorter and the normal segments are elongated and joined by the insertion of exogenous nucleotides to form high molecular weight DNA as in nonirradiated cells. These processes occur in normal human cells, where UV-induced pyrimidine dimers are excised, as well as in xeroderma pigmentosum (XP) cells, where dimers are not excised. The effect of caffeine on these processes was determined for both normal human and XP cells. Caffeine, which binds to denatured regions of DNA, inhibited DNA chain elongation and joining in irradiated XP cells but not in irradiated normal human or nonirradiated cells. Caffeine also caused an alteration in the ability to recover synthesis of DNA of normal size at long times after irradiation in XP cells but not in normal cells.     

Postreplication repair of deoxyribonucleic acid and daughter strand exchange in uvr- mutants of Bacillus subtilis

The fate of pyrimidine dimers in deoxyribonucleic acid (DNA) newly synthesized by Bacillus subtilis after ultraviolet irradiation was monitored by use of a damage-specific endonuclease that introduces single-strand breaks adjacent to nearly all of the dimer sites. Two Uvr- strains, one defective in the initiation of dimer excision and the other defective in a function required for efficient dimer excision, were found to be similar to their wild-type parent in the kinetics and extent of converting low-molecular-weight DNA newly synthesized after ultraviolet irradiation to high molecular weight. In the Uvr- strains large molecules of newly synthesized DNA remained susceptible to nicking by the damage-specific endonuclease even after extended incubation in growth medium, whereas the enzyme-sensitive sites were rapidly removed from both preexisting and newly synthesized DNA in Uvr+ cells. Our results support the hypothesis that postreplication repair in bacteria includes recombination between dimer-containing parental DNA strands and newly synthesized strands.     

衰老过程中大白鼠脾细胞的DNA单链断裂与重接能力的变化

 DNA被紫外线损伤后,由DNA切除修复酶切除嘧啶二聚体,随之以另一条正常的DNA链为模板修复合成DNA片段,最后由DNA连接酶将新合成的DNA片与原有的DNA链连接。本文用荧光法测定DNA修复过程中DNA单链的断裂及重接能力与衰老的关系。结果表明,不同年龄大鼠脾细胞均具有修复DNA单链断裂的能力,DNA单链断裂重接的能力与年龄有相关性,断乳鼠及青年鼠的脾细胞当保温至30min时,即开始了DNA链的重接,保温90min后则恢复到原有水平;而老年鼠脾细胞保温至90min时才开始DNA链的重接,保温150min,尚未恢复到原有水平。还发现,断乳鼠及老年鼠脾细胞的单链DNA含量高于青年鼠。     

Inhibition of DNA replication by hydroxyurea and caffeine in an ultraviolet-irradiated human fibroblast cell line

DNA replication in human fibroblasts with normal excision repair was investigated after ultraviolet irradiation and incubation with caffeine or hydroxyurea. The DNA synthesized soon after irradiation had a reduced size, but that synthesized later was near normal size. When caffeine was present before labeling, it reduced the size of DNA synthesized but when added after labeling it was without effect. When irradiated cells were allowed to grow, labeled DNA increased in size steadily for 60 min to a maximum that was below control and dose-dependent. Further growth resulted in a transition of some label to parental DNA sizes, but a large fraction remained permanently blocked at smaller sizes producing bimodal distributions of DNA. The steady increase in size was inhibited by hydroxyurea. Removing cells from hydroxyurea resulted in increases similar to or slightly slower than those observed immediately after labeling, and this protocol did not permit cells to acquire any induced or enhanced capacity to replicate damaged DNA.     

Ligase-deficient yeast cells exhibit defective DNA rejoining and enhanced gamma ray sensitivity.

Yeast cells deficient in DNA ligase were also deficient in their capacity to rejoin single-strand scissions in prelabeled nuclear DNA. After high-dose-rate gamma irradiation (10 and 25 krads), cdc9-9 mutant cells failed to rejoin single-strand scissions at the restrictive temperature of 37 degrees C. In contrast, parental (CDC9) cells (incubated with mutant cells both during and after irradiation) exhibited rapid medium-independent DNA rejoining after 10 min of post-irradiation incubation and slower rates of rejoining after longer incubation. Parental cells were also more resistant than mutant cells to killing by gamma irradiation. Approximately 2.5 +/- 0.07 and 5.7 +/- 0.6 single-strand breaks per 10(8) daltons were detected in DNAs from either CDC9 or cdc9-9 cells converted to spheroplasts immediately after 10 and 25 krads of irradiation, respectively. At the permissive temperature of 23 degrees C, the cdc9-9 cells contained 2 to 3 times the number of DNA single-strand breaks as parental cells after 10 min to 4 h of incubation after 10 krads of irradiation, and two- to eightfold more breaks after 10 min to 2.5 h of incubation after 25 krads of irradiation. Rejoining of single-strand scissions was faster in medium. After only 10 min in buffered growth medium and after 10 krads of irradiation, the number of DNA single-strand breaks was reduced to 0.32 +/- 0.3 (at 23 degrees C) or 0.21 +/- 0.05 (at 37 degrees C) per 10(8) daltons in parental cells, but remained at 2.1 +/- 0.06 (at 23 degrees C) or 2.3 +/- 0.07 (at 37 degrees C) per 10(8) daltons in mutant cells. After 10 or 25 krads of irradiation plus 1 h of incubation in medium at 37 degrees C, only DNA from CDC9 cells was rejoined to the size of DNA from unirradiated cells, whereas at 23 degrees C, DNAs in both strains were completely rejoined.     

Repair of gamma-ray induced DNA strand breaks in the radiation-sensitive mutant rad18-2 of Saccharomyces cerevisiae

The repair of gamma-ray induced DNA single and double-strand breaks was looked at in wild type and rad18-2 strains of the yeast Saccharomyces cerevisiae using sucrose gradient centrifugation. It was found that rad18-2 diploid cells could repair single and double-strand breaks induced by gamma-rays. It was also found that rad18-2 cells experienced a breakup of their DNA during post-irradiation incubation to a size smaller than seen in cells just receiving irradiation. This breakup of DNA in rad18-2 cells is not degradation due to cell death since wild type cells irradiated to similar low survival levels do not show this breakup of DNA with 8 h incubation. The breakup of DNA in rad18-2 cells is not due to replication gaps being formed by synthesis on a damaged template since treatment of rad18-2 a mating type cells with alpha factor, to prevent initiation of DNA synthesis, does not prevent breakup of the DNA.     

Effect of divalent ions on the minimal relaxase domain of MobA

The MobA protein encoded by plasmid R1162 plays an important role in conjugative mobilization between bacterial cells. It has two functional domains, the N-terminal relaxase domain and C-terminal primase domain. The N-terminal 186 residues (minMobA) is the minimal domain required for relaxase activity. We investigated the effects of different divalent metallic cations on minMobA activity measuring DNA binding, DNA nicking, and protein denaturation experiments. The results show that divalent cations are not required for DNA binding but are required for DNA nicking. The range of metals that function in minMobA suggests the cation role is largely structural. The most tightly binding cation is Mn²⁺, but the expressed protein shows roughly equal amounts of Mg²⁺ and Ca²⁺, both of which facilitate substrate binding and catalysis. Surprisingly, Zn²⁺ does not facilitate DNA binding nor allow nicking activity.     

Synthesis and DNA nicking studies of a novel cyclic peptide: cyclo[Lys-Trp-Lys-Ahx-].

Two novel cyclic tetrapeptides: cyclo[Lys-Tyr-Lys-Ahx-] 7a and cyclo[Lys-Trp-Lys-Ahx-] 7b were synthesized by coupling protected amino acid in solution and the subsequent cyclization effected by the pentafluorophenyl ester method as described in previous papers. These cyclic peptides were designed and synthesized to study their interaction with DNA, based on previous reports that linear peptides Lys-Tyr-Lys and Lys-Trp-Lys could bind to various forms of DNA and cleaved supercoiled DNA at apurinic sites. Ethidium bromide displacement assay showed that the apparent DNA binding constant of linear Lys-Tyr-Lys and cyclic peptide 7a are far below 1 x 10(3) M(-1), whereas those of cyclic peptide 7b and linear Lys-Trp-Lys are 1.9 x 10(4) M(-1) and 9.5 x 10(3) M(-1), respectively. Kinetic studies using agarose gel electrophoresis showed that cyclic peptide 7b and Lys-Trp-Lys possessed DNA nicking activity on natural supercoiled phi X174 DNA with nicking rate of 50.7 and 75.6 pM min(-1) at 65 degrees C, respectively, whereas cyclic peptide 7a and linear Lys-Tyr-Lys were devoid of the corresponding activity. The DNA nicking rate increased significantly with increase in reaction temperature. At reaction temperatures lower than 65 degrees C, the DNA nicking rate of cyclic peptide 7b exceeded that of linear Lys-Trp-Lys. The addition of 1 microM ferrous ion did not give significant enhancement effect on the DNA nicking rate by the peptides. UV irradiation gave a marked rate enhancement on the DNA nicking rate of linear Lys-Trp-Lys and a moderate enhancement on the DNA nicking rate of cyclic peptide 7b.     

Mechanism of gap-filling during postreplication repair of ultraviolet damage in Haemophilus influenzae.

Deoxyribonucleic acid (DNA), pulse labeled after ultraviolet irradiation of excision-defective mutants of Haemophilus influenzae, is of lower single strand molecular weight than that of unirradiated cells but approaches the size of DNA from unirradiated cells upon further incubation in growth medium. This gap-filling process is controlled by the rec-1 gene. Gap-filling occurs normally in a temperature-sensitive DNA synthesis mutant at the restrictive temperature showing that normal semiconservative DNA synthesis is not necessary for gap-filling. To test for recombinational events after irradiation, the DNA synthesized after irradiation was radioactively labeled for a short time in medium containing 5-bromodeoxyuridine followed by incubation for various times in non-radioactive, 5-bromodeoxyuridine-containing medium. The DNA was denatured and analyzed isopycnically. The labeled DNA was initially "heavy," but later shifted toward lighter densities. This shift occurred in the temperature-sensitive DNA synthesis mutant at the restrictive temperature and in the recombination-defective mutant rec-2, but was not seen in the rec-1 mutant. The density shift can be interpreted as evidence that rather extensive exchanges occurred between parental DNA and the DNA made after irradiation. These results suggest that such exchanges are important for gap-filling in H. influenzae.     

Genetic control of excision of Saccharomyces cerevisiae interstrand DNA cross-links induced by psoralen plus near-UV light.

Excision of interstrand DNA cross-links induced by 4,5',8-trimethyl psoralen plus 360-nm light was examined in wild type (RAD+) and various radiation-sensitive (rad) mutants of Saccharomyces cerevisiae known to be defective in the excision of UV light-induced pyrimidine dimers. Alkaline sucrose sedimentation of DNA after incubation of psoralen-plus-light-treated cells indicated little or no nicking of cross-linked DNA in rad1-2, rad2-5, rad3-2, rad4-4, rad10-2, and mms19-1 mutants. In the rad14-2 mutant, substantial nicking was observed but to a much lesser extent than in the RAD+ strains, whereas the rad16-1 mutant was as proficient in nicking as the RAD+ strain. Removal of cross-links was also examined in RAD+, rad3-2, and rad14-2 strains by determining the sensitivity of alkali-denatured and -neutralized DNA to hydrolysis by S1 nuclease. No cross-link removal was observed in the rad3-2 mutants, and the rad14-2 mutant was much less efficient than the RAD+ strain in removing cross-links.     

Gaps in DNA synthesized by ultraviolet light-irradiated WI38 human cells.

DNA replication in ultraviolet-irradiated human cells was examined by treatment of the extracted DNA with a single-strand specific endonuclease from Neurospora crassa. WI38 cells were uniformly labeled with 32Pi for two generations before irradiation and then labeled with [3H]thymidine after irradiation. The isolated DNA was sedimented in neutral sucrose gradients after incubation with the endonuclease. The endonuclease treatment had no effect on the sedimentation profiles of either [32P]DNA or [3H]DNA from unirradiated control cultures. The endonuclease treatment also did not significantly alter the profile of [32P]DNA from irradiated cultures but did introduce breaks in the 3H pulse-labeled DNA synthesized after irradiation. These results indicate that DNA synthesis after ultraviolet irradiation proceeds in such fashion that gaps are formed along the newly made strand, leaving regions of single strandness in template DNA. As replication proceeds these gaps disappear and 2 h after irradiation (100-250 ergs/mm2) they are barely detectable by the endonuclease assay.     

Psoralen cross-linking as probe of torsional tension and topological domain size in vivo

DNA within a cell is organized with unrestrained torsional tension, and each molecule is divided into multiple individual topological domains. Psoralen photobinding can be used as an assay for supercoiling and topological domain size in living cells. Psoralen photobinds to DNA at a rate nearly linearly proportional to superhelical density. Comparison of the rate of photobinding to supercoiled and relaxed DNA in cells provides a measure of superhelical density. For this, in vivo superhelical tension is relaxed by the introduction of nicks by either ionizing radiation or photolysis of bromodeoxyuridine in the DNA. Since nicks are introduced in a random fashion, the distribution of nicks is described by a Poisson distribution. Thus, after nicking, the fraction of topological domains containing no nicks is described by the zero term of the Poisson distribution. From measurement of the number of nicks introduced in the DNA and the fraction of torsional tension remaining, an average topological domain size can be estimated. Using this logic, procedures were designed and described for measuring supercoiling and domain size at specific sites in eukaryotic genomes.     

A specific DNA unwinding activity associated with SV40 large T antigen

The incubation of highly purified large T antigen with relaxed, circular SV40 DNA in the presence of topoisomerase I (nicking closing enzyme) resulted in the introduction of negative superhelical turns in the DNA. ATP was not required for this reaction. A similar introduction of superhelical turns could also be obtained when a recombinant plasmid DNA (Y182), which contains sequences from both SV40 DNA and pBR322, was used. However, no effect was observed when relaxed pBR322 DNA, which does not contain SV40 DNA sequences, was incubated with T antigen in the presence of topoisomerase. These results are consistent with the hypothesis that large T antigen can recognize and unwind specific sequences on SV40 DNA.     

A comparison of the in vitro genotoxicity of anticancer drugs idarubicin and mitoxantrone

Idarubicin is an anthracycline antibiotic used in cancer therapy. Mitoxantrone is an anthracycline analog with presumed better antineoplastic activity and lesser toxicity. Using the alkaline comet assaywe showed that the drugs at 0.01-10 microM induced DNA damage in normal human lymphocytes. The effect induced by idarubicin was more pronounced than by mitoxantrone (P < 0.001). The cells treated with mitoxantrone at 1 microM were able to repair damage to their DNA within a 30-min incubation, whereas the lymphocytes exposed to idarubicin needed 180 min. Since anthracyclines are known to produce free radicals, we checked whether reactive oxygen species might be involved in the observed DNA damage. Catalase, an enzyme inactivating hydrogen peroxide, decreased the extent of DNA damage induced by idarubicin, but did not affect the extent evoked by mitoxantrone. Lymphocytes exposed to the drugs and treated with endonuclease III or formamidopyrimidine-DNA glycosylase (Fpg), enzymes recognizing and nicking oxidized bases, displayed a higher level of DNA damage than the untreated ones. 3-Methyladenine-DNA glycosylase II (AlkA), an enzyme recognizing and nicking mainly methylated bases in DNA, increased the extent of DNA damage caused by idarubicin, but not that induced by mitoxantrone. Our results indicate that the induction of secondary malignancies should be taken into account as side effects of the two drugs. Direct strand breaks, oxidation and methylation of the DNA bases can underlie the DNA-damaging effect of idarubicin, whereas mitoxantrone can induce strand breaks and modification of the bases, including oxidation. The observed in normal lymphocytes much lesser genotoxicity of mitoxantrone compared to idarubicin should be taken into account in planning chemotherapeutic strategies.     

UV-A induced DNA nicking activities of skin photosensitive drugs: phenothiazines, benzothiadiazines and afloqualone

Plasmid pBR 322 was subjected to UV-A irradiation in the presence of photosensitive drugs, i.e., phenothiazines [chlorpromazine hydrochloride (CPZ), promethazine hydrochloride (PMZ) and mequitazine (MQZ)], benzothiadiazines [penflutizide (PFZ), hydrochlorothiazide (HCT) and methyclothiazide (MCT)] and afloqualone (AQ). The distribution of the closed-circular and the open-circular form of the plasmid DNA was analyzed by means of neutral agarose gel electrophoresis. All the drugs used induced more or less DNA nicking to yield the open-circular form. The nicking activities of the phenothiazines were in the order: CPZ greater than PMZ greater than MQZ. CPZ elicited extensive degradation of the DNA by photosensitization. The nicking activities of the benzothiadiazines and AQ were much weaker than CPZ and PMZ.     

Increased Resistance to the Spread of Tobacco Mosaic Virus in Pinto Bean Leaves Caused by Sugar and Light

Ultraviolet light (UV) irradiation increased expansion of TMV lesions in detached Pinto bean primary leaves incubated in darkness. However, if after UV-irradiation the leaves were incubated in the light, no increase in lesion expansion occurred. The light effect was considered not to be due to photorepair of UV damaged DNA, since non-photorepairing treatments such as incubation in red light, or delayed exposure to white light after UV irradiation also prevented increase in lesion expansion. The effect of visible light in preventing TMV-lesion enlargement was shown to be related to photosynthetic energy supply to the host cell defense mechanism since incubation of infected leaves in the presence of the photosynthesis inhibitor 3-(3,4-dichlorophenyl)-l,l-dimethyl urea (DCMU) in light caused large lesions whether leaves were irradiated by UV or not. Supplying 0.1 M sucrose in the dark also inhibited lesion enlargement in UV-irradiated or nonirradiated leaves. Dinitrophenol (DNP) negated the sucrose effect in the dark. However, in light incubation, DNP did not induce large lesions indicating that DNP did not interfere with energy supply in the light. It is concluded that the Pinto bean leaf cells can use energy derived both from mitochondria and chloroplasts for building the resistance mechanism to virus spread. In this case, cellular resistance to virus spread seems to be correlated with callose deposition on the walls of noninfected cells adjacent to the necrotic cells. Energy supply in various forms will assist host cells in building the resistance mechanism as well as retarding senescence. Detachment, prolonged dark incubation, or exogenous supply of DNP led to accelerated senescence which in turn led to secondary enlargement of lesions. The cause of such secondary enlargement may be explained by starvation of cells and disappearance of callose.     

DNA repair in Proteus mirabilis. IV. Post-irradiation DNA degradation as influenced by a function inducible in rec+ cells.

Summary Post-irradiation DNA degradation in P. mirabilis rec ⁺ strains after UV irradiation is found to be more extensive in starvation buffer than in growth medium. In growth medium restriction of protein synthesis, but not DNA synthesis, largely prevents the expression of breakdown limitation. By the addition of chloramphenicol during post-irradiation incubation in growth medium the expression of break-down limitation was followed and found to occur 20 to 40 min after UV irradiation. Pre-irradiation by a low dose of UV leads after a corresponding time of post-irradiation incubation to breakdown limitation even in starvation buffer after a second UV exposure.Post-irradiation DNA degradation is presumed to be initiated at the sites of DNA lesions which arise at replication points damaged by UV. While pre-starvation restricts the efficiency of postirradiation DNA degradation by the reduction of the number of replication points active at the time of irradiation, caffeine as well as 2,4-dinitrophenol inhibit DNA degradation even in rec ^- cells probably by the interference with nicking or exonucleoltytic events initiated at those sites in the absence of breakdown limitation.Breakdown limitation is postulated to be due to inducible derepression of REC-functions which lead to the protection and, probably, repair of DNA lesions arising at the replication points following UV exposure.     

Tungsten particle-induced nicking of supercoiled plasmid DNA

Small particles of metallic tungsten, known also as tungsten microprojectiles, are routinely used for biotechnological purposes. In such applications, tungsten was observed to affect the integrity of plasmid DNA. Here we present evidence that interaction between tungsten particles and intact circular plasmids pU19, pUC119, and ColE1 may result in generation of a limited number of single-strand DNA breaks. As a consequence, supercoiled DNA is converted into its open circular form and no fragmentation products can be detected. The rate of the tungsten-mediated reaction depends on pH but is not influenced by ascorbate, Tris, or EDTA. No DNA nicking can be observed when the tungsten particles are replaced by substances that can be leached out from these particles with water or incubation buffers. Likewise, commercial sodium tungstate, tungsten (VI) oxide, and tungsten (VI) chloride and products of its decomposition remain DNA undamaged. Native plasmid DNA molecules, upon adsorption on the surface of tungsten microparticles, may undergo some nicking without a need for participation of external catalysts.     

DNA Repair in Potorous tridactylus

The DNA synthesized shortly after ultraviolet (UV) irradiation of Potorous tridactylis (PtK) cells sediments more slowly in alkali than that made by nonirradiated cells. The size of the single-strand segments is approximately equal to the average distance between 1 or 2 cyclobutyl pyrimidine dimers in the parental DNA. These data support the notion that dimers are the photoproducts which interrupt normal DNA replication. Upon incubation of irradiated cells the small segments are enlarged to form high molecular weight DNA as in nonirradiated cells. DNA synthesized at long times (~ 24 h) after irradiation is made in segments approximately equal to those synthesized by nonirradiated cells, although only 10-15% of the dimers have been removed by excision repair. These data imply that dimers are not the lesions which initially interrupt normal DNA replication in irradiated cells. In an attempt to resolve these conflicting interpretations, PtK cells were exposed to photoreactivating light after irradiation and before pulse-labeling, since photoreactivation repair is specific for only one type of UV lesion. After 1 h of exposure ~ 35% of the pyrimidine dimers have been monomerized, and the reduction in the percentage of dimers correlates with an increased size for the DNA synthesized by irradiated cells. Therefore, we conclude that the dimers are the lesions which initially interrupt DNA replication in irradiated PtK cells. The monomerization of pyrimidine dimers correlates with a disappearance of repair endonuclease-sensitive sites, as measured in vivo immediately after 1 h of photoreactivation, indicating that some of the sites sensitive to the repair endonuclease (from Micrococcus luteus) are pyrimidine dimers. However, at 24 h after irradiation and 1 h of photoreactivation there are no endonuclease-sensitive sites, even though ~ 50% of the pyrimidine dimers remain in the DNA. These data indicate that not all pyrimidine dimers are accessible to the repair endonuclease. The observation that at long times after irradiation DNA is made in segments equal to those synthesized by nonirradiated cells although only a small percentage of the dimers have been removed suggests that an additional repair system alters dimers so that they no longer interrupt DNA replication.