Alkaline Sucrose Density Gradient Research Articles

A large intermediate domain of vertebrate REV3 protein is dispensable for ultraviolet-induced translesion replication

DNA polymerase ζ (pol ζ) is involved in translesion replication (translesion synthesis, TLS) and plays an essential role in embryogenesis. In adults, pol ζ triggers mutation as a result of error-prone TLS and causes carcinogenesis. The catalytic subunit of pol ζ, REV3, is evolutionarily conserved from yeast and plants to higher eukaryotes. However, the structures are notably different: unlike that in yeast REV3, a large intermediate domain is inserted in REV3 of humans and mice. The domain is mostly occupied with noncommittal structures (random coil…etc.); therefore, its role and function are yet to be resolved. Previously, we reported deficient levels of ultraviolet (UV)-induced TLS in fibroblasts derived from the Rev3-knockout mouse embryo (Rev3KO-MEF). Here, we constructed a mouse Rev3-expressing plasmid with a deleted intermediate domain (532-1793 a.a,) and transfected it into Rev3KO-MEF. The isolated stable transformants showed comparable levels of UV-sensitivity and UV-TLS activity to those in wild-type MEF, detected using an alkaline sucrose density gradient sedimentation. These results indicate that the intermediate domain is nonessential for UV-induced translesion replication in cultured mouse cells.

Trapping DNA Replication Origins from the Human Genome

Synthesis of chromosomal DNA is initiated from multiple origins of replication in higher eukaryotes; however, little is known about these origins’ structures. We isolated the origin-derived nascent DNAs from a human repair-deficient cell line by blocking the replication forks near the origins using two different origin-trapping methods (i.e., UV- or chemical crosslinker-treatment and cell synchronization in early S phase using DNA replication inhibitors). Single-stranded DNAs (of 0.5–3 kb) that accumulated after such treatments were labeled with bromodeoxyuridine (BrdU). BrdU-labeled DNA was immunopurified after fractionation by alkaline sucrose density gradient centrifugation and cloned by complementary-strand synthesis and PCR amplification. Competitive PCR revealed an increased abundance of DNA derived from known replication origins (c-myc and lamin B2 genes) in the nascent DNA fractions from the UV-treated or crosslinked cells. Nucleotide sequences of 85 and 208 kb were obtained from the two libraries (I and II) prepared from the UV-treated log-phase cells and early S phase arrested cells, respectively. The libraries differed from each other in their G+C composition and replication-related motif contents, suggesting that differences existed between the origin fragments isolated by the two different origin-trapping methods. The replication activities for seven out of 12 putative origin loci from the early-S phase cells were shown by competitive PCR. We mapped 117 (library I) and 172 (library II) putative origin loci to the human genome; approximately 60% and 50% of these loci were assigned to the G-band and intragenic regions, respectively. Analyses of the flanking sequences of the mapped loci suggested that the putative origin loci tended to associate with genes (including conserved sites) and DNase I hypersensitive sites; however, poor correlations were found between such loci and the CpG islands, transcription start sites, and K27-acetylated histone H3 peaks.

Caffeine Abolishes the Ultraviolet-Induced REV3 Translesion Replication Pathway in Mouse Cells

When a replicative DNA polymerase stalls upon encountering a photoproduct on the template strand, it is relieved by other low-processivity polymerase(s), which insert nucleotide(s) opposite the lesion. Using an alkaline sucrose density gradient sedimentation technique, we previously classified this process termed UV-induced translesion replication (UV-TLS) into two types. In human cancer cells or xeroderma pigmentosum variant (XP-V) cells, UV-TLS was inhibited by caffeine or proteasome inhibitors. However, in normal human cells, the process was insensitive to these reagents. Reportedly, in yeast or mammalian cells, REV3 protein (a catalytic subunit of DNA polymerase ζ) is predominantly involved in the former type of TLS. Here, we studied UV-TLS in fibroblasts derived from the Rev3-knockout mouse embryo (Rev3KO-MEF). In the wild-type MEF, UV-TLS was slow (similar to that of human cancer cells or XP-V cells), and was abolished by caffeine or MG-262. In 2 cell lines of Rev3KO-MEF (Rev3−/− p53−/−), UV-TLS was not observed. In p53KO-MEF, which is a strict control for Rev3KO-MEF, the UV-TLS response was similar to that of the wild-type. Introduction of the Rev3 expression plasmid into Rev3KO-MEF restored the UV-TLS response in selected stable transformants. In some transformants, viability to UV was the same as that in the wild-type, and the death rate was increased by caffeine. Our findings indicate that REV3 is predominantly involved in UV-TLS in mouse cells, and that the REV3 translesion pathway is suppressed by caffeine or proteasome inhibitors.

Rev1, Rev3, or Rev7 siRNA Abolishes Ultraviolet Light‐Induced Translesion Replication in HeLa Cells: A Comprehensive Study Using Alkaline Sucrose Density Gradient Sedimentation

When a replicative DNA polymerase stalls upon encountering a lesion on the template strand, it is relieved by other low-processivity polymerase(s), which insert nucleotide(s) opposite the lesion, extend by a few nucleotides, and dissociate from the 3′-OH. The replicative polymerase then resumes DNA synthesis. This process, termed translesion replication (TLS) or replicative bypass, may involve at least five different polymerases in mammals, although the participating polymerases and their roles have not been entirely characterized. Using siRNAs originally designed and an alkaline sucrose density gradient sedimentation technique, we verified the involvement of several polymerases in ultraviolet (UV) light-induced TLS in HeLa cells. First, siRNAs to Rev3 or Rev7 largely abolished UV-TLS, suggesting that these 2 gene products, which comprise Polζ, play a main role in mutagenic TLS. Second, Rev1-targeted siRNA also abrogated UV-TLS, indicating that Rev1 is also indispensable to mutagenic TLS. Third, Polη-targeted siRNA also prevented TLS to a greater extent than our expectations. Forth, although siRNA to Polι had no detectable effect, that to Polκ delayed UV-TLS. To our knowledge, this is the first study reporting apparent evidence for the participation of Polκ in UV-TLS.

A novel Rad18 function involved in protection of the vertebrate genome after exposure to camptothecin

In Saccharomyces cerevisiae, Rad18 functions in post-replication repair pathways, such as error-free damage bypass involving Rad30 (Polη) and error-prone damage bypass involving Rev3/7 (Polζ). Chicken DT40 RAD18 −/− cells were found to be hypersensitive to camptothecin (CPT), while RAD30 −/− and REV3 −/− cells, which are defective in translesion DNA synthesis, were not. RAD18 −/− cells also showed higher levels of H2AX phosphorylation and chromosomal aberrations, particularly chromosomal gaps and breaks, upon exposure to CPT. Detailed analysis by alkaline sucrose density gradient centrifugation revealed that RAD18 −/− and wild type cells exhibited similar rates of elongation of newly synthesized DNA in the presence or absence of low concentrations of CPT but that DNA breaks frequently occurred on both parental and nascent strands within 1 h after a brief exposure to an elevated concentration of CPT, with more breaks induced in RAD18 −/− cells than in wild type cells. These data suggest a previously unanticipated role for Rad18 in dealing with replication forks upon encountering DNA lesions induced by CPT.

Translesion replication in cisplatin-treated xeroderma pigmentosum variant cells is also caffeine-sensitive: features of the error-prone DNA polymerase(s) involved in UV-mutagenesis

Patients with xeroderma pigmentosum variant (XP-V) have a higher risk to skin cancer and XP-V cells are extremely mutable by ultraviolet (UV). The defective gene encodes a DNA polymerase (Polη) which catalyzed relatively accurate translesion synthesis past the cyclobutane dimer of UV-lesions instead of the replicative polymerase(s) that stalled just before the lesion. Pulse-chase studies have shown that translesion replication in XP-V cells is delayed, but does not completely cease. Taking these results together, error-prone polymerase(s) are plausively involved in the UV-mutagenesis in XP-V devoid of Polη. However, less is known about the polymerase(s) in vivo. Using an alkaline sucrose density gradient centrifugation (ASDG) technique, translesion replication is detected in the two XP-V strains XP30RO and XP115LO. As reported by Lehmann et al. [Proc. Natl. Acad. Sci. U.S.A. 72 (1975): 219] in XP-V; (i) smaller replication products were accumulated after UV irradiation; (ii) the elongation of these products was delayed; (iii) the elongation was markedly inhibited by caffeine. XP-V cells UV-irradiated at mid-S phase were normally S-arrested, and no “override” by caffeine (i.e. abrogation of the S-checkpoint) was observed by flow cytometry, suggesting that caffeine does not act via cdc kinase here; (iv) butylphenyldeoxyguanosine (BuPGdR) inhibited elongation of replication products only in UV-irradiated XP-V cells; (v) dideoxycytidine or dideoxyinosine had no effect on this process in either normal or XP-V cells. Next, similar phenomena to UV (all of above i to v) were observed also in cisplatin-treated XP-V cells. Polη was indicated to participate in cisplatin-induced translesion replication in normal cells. Summing up the above results, the polymerase(s) which work in translesion replication in XP-V are probably BuPGdR-sensitive, insensitive to dideoxynucleotides and can bypass also cisplatin-lesions. To date, several polymerases capable of lesion-bypass synthesis have been isolated. The features presented here are quite useful for identifying the error-prone polymerase(s) involved in UV-mutagenesis.

Damage to DNA and activity of nuclear DNA repair and replicative enzymes following N‐nitrosodiethylamine treatment to rats

Continuous administration in the drinking water of hepatocarcinogen N-nitrosodiethylamine (NDEA) to male rats (200 mg/L) for 60 days resulted in DNA damage in the form of single strand breaks. The damage, which is measured as a shift in the sedimentation of DNA in alkaline sucrose density gradients, was found to be maximum at the fourth week of treatment, and the sedimentation pattern of DNA was found to return to near normal size by the seventh week of NDEA treatment. Simultaneously, there were perturbations in the nuclear enzymes involved in DNA replication and repair. Activities of DNA polymerase β, DNA ligase, and topoisomerase were found to increase in as early as the first week of NDEA treatment and reached the maximum at the fourth week, and thereafter declined to normal level by the eighth week of treatment. Concomitantly, the activities of DNA polymerase α, DNA primase, and RNA polymerase which were unaltered in the initial period of carcinogen treatment recorded a marked increase after sixth week of NDEA treatment. Results suggest that administration of NDEA inflicts DNA damage, which is manifested as increase in DNA repair enzymes in the initial period and activated DNA replicative enzymes at a later period, indicating the active proliferation of transformed cells. © 2000 John Wiley & Sons, Inc. J Biochem Toxicol 14:277–282, 2000

Damage to DNA and activity of nuclear DNA repair and replicative enzymes following N-nitrosodiethylamine treatment to rats.

Continuous administration in the drinking water of hepatocarcinogen N-nitrosodiethylamine (NDEA) to male rats (200 mg/L) for 60 days resulted in DNA damage in the form of single strand breaks. The damage, which is measured as a shift in the sedimentation of DNA in alkaline sucrose density gradients, was found to be maximum at the fourth week of treatment, and the sedimentation pattern of DNA was found to return to near normal size by the seventh week of NDEA treatment. Simultaneously, there were perturbations in the nuclear enzymes involved in DNA replication and repair. Activities of DNA polymerase beta, DNA ligase, and topoisomerase were found to increase in as early as the first week of NDEA treatment and reached the maximum at the fourth week, and thereafter declined to normal level by the eighth week of treatment. Concomitantly, the activities of DNA polymerase alpha, DNA primase, and RNA polymerase which were unaltered in the initial period of carcinogen treatment recorded a marked increase after sixth week of NDEA treatment. Results suggest that administration of NDEA inflicts DNA damage, which is manifested as increase in DNA repair enzymes in the initial period and activated DNA replicative enzymes at a later period, indicating the active proliferation of transformed cells.

Alkaline Density Gradient Floatation of Membranes: Polypeptide Composition of the Mammalian Peroxisomal Membrane

A method for purification of the peroxisomal membrane from rat liver is described. The procedure consists of floating the (contaminated) peroxisomal membranes through an alkaline sucrose density gradient. A good resolution between the peroxisomal membrane and other membrane systems is achieved. Using these floated peroxisomal membranes we have determined that only 7.8 ± 0.9% of the total peroxisomal protein is alkali resistant. The polypeptide composition of these highly pure peroxisomal membranes was analyzed by SDS–PAGE. Bands corresponding to polypeptides with apparent molecular masses of 15, 18, 22, 24, 26, 29, 35, 36, 38, 40, 52, 55, 70, 74–77, and 88 kDa are detected upon Coomassie blue staining of polyacrylamide gels. The identity of several of these polypeptides was determined by N-terminal sequencing and Western blotting analysis.

Immunochemical detection of oxidative DNA damage in cancer and aging using anti-reactive oxygen species modified DNA monoclonal antibody

The formation of reactive oxygen species (ROS), although a normal cellular activity, is considerably enhanced under chronic inflammatory conditions and ischemia. These species have been implicated in various disorders, mutagenesis, carcinogenesis and aging. Of many macromolecules, DNA is the most susceptible to hydroxyl radical, the most reactive of the ROS. The present study is designed to detect oxidative DNA damage in cancer patients and healthy aged humans using an anti-ROS-DNA monoclonal antibody (mAb). Purified calf thymus DNA fragments (approximate size 400 bp) were modified with ·OH, generated by UV-irradiation (254 nm) of hydrogen peroxide. ROS-modified DNA was characterized by UV-spectroscopy, melting temperature, alkaline sucrose density gradient ultracentrifugation and ion-exchange chromatography. ROS-DNA showed single strand breaks, decrease in T m, modification of thymine (58.3%) and guanine (20%). The mAb generated against ROS-DNA was characterized for antigen binding specificity by competition ELISA. Monoclonal antibody showed strong binding to ROS-modified DNA, its modified fragments, polynucleotides and bases. With the exception of native DNA, binding of unmodified polynucleotides and bases was much lower. The mAb distinctly recognized DNA samples from lymphocytes of healthy aged humans and gave maximum inhibitions of 49, 53, 64 and 70%, while not reacting with DNA from young population. Similarly, oxidative lesions in DNA from cancer patients were also efficiently recognized by the mAb. DNA from healthy controls served as negative control. The studies demonstrate that the mAb, although cross-reactive, preferentially binds ROS-modified epitopes on DNA. High reactivity of mAb to DNA samples from cancer patients and healthy aged humans indicates increased oxidative stress leading to DNA damage.

An improved method of alkaline sucrose density gradient sedimentation to detect less than one lesion per 1 Mb DNA.

We improved alkaline sucrose density gradient sedimentation to detect very long single-strand DNA at the megabase level (from less than 1 to about 4 Mb). Hitherto, these have not sedimented correctly due to some artifacts. One artifact was aggregation of sticky DNA and proteins formed in the gradient. Then, in some gradients, biphasic distribution was observed, the major peak of which was reasonable as a result of random scission by X-rays, but the minor, fast-sedimenting population was another artifact resulting from incomplete denaturation of the DNA. We mainly reduced the centrifugal force and used a solution for cell lysis with a high concentration of salt. By means of this procedure, DNA single-strand breaks induced by relatively low doses of X-rays and subsequent repair processes can be measured in human fibroblasts. The protocol is also applicable to the study of DNA damage accompanied by strand scission, such as by UV or dimethyl sulfate as well as their repair. The technique is sensitive enough to detect even single-strand breaks induced by 0.1 J/m2 UV and sufficiently reproducible that breaks induced by increasing UV dosages were dose dependent. Thus, this technique was proven to be very sensitive, reliable and simple to perform. Therefore, this improvement will be extremely useful to investigators studying DNA repair.

Effect of glutathione on lambda deoxyribonucleic acid strand breaks in the reaction system of glutathione-alloxan in the presence of Fe(3+)-ethylenediaminetetraacetic acid.

Alkaline sucrose density gradient and agarose gel electrophoresis methods were used to observe lambda deoxyribonucleic acid (DNA) strand breaks by the reaction system of reduced glutathione (GSH) with alloxan in the presence of Fe(3+)-ethylenediaminetetraacetic acid (EDTA). When DNA was incubated in the reaction system for 10 min, DNA strand breaks were easily induced. The increasing concentrations of GSH up to 1.0 mM in the reaction system in the presence of 1.0 mM alloxan caused DNA strand breaks in a concentration-dependent fashion and GSH beyond 2.0 mM caused in the strand breaks of DNA by which the fragments with multiple ranges of molecular weight were produced. The strand breaks of DNA in the reaction system containing low concentrations of GSH were protected by catalase and hydroxyl radical (HO.) scavengers but superoxide dismutase (SOD) did not, indicating that such breaks were induced by HO.generated from the Fenton reaction. On the other hand, the strand breaks of DNA at high concentrations of GSH were protected by ethanol and desferrioxamine, but not effectively by SOD and HO.scavengers, suggesting the possible participation of some oxidizing species of iron rather than HO.. These results indicate that HO.or oxidizing species of iron generated in the GSH-alloxan system depending on the concentration of GSH attacks DNA to produce strand breaks.

Effect of ultraviolet light on DNA structure in 5-iodo-2′-deoxyuridine-substituted HSV-1 DNA

Herpes simplex virus type-1 (HSV-1) was grown in the presence of 5-iodo-2′-deoxyuridine (IdUrd), and the virion-DNA was isolated by isopycnic centrifugation in CsCl. Irradiation of IdUrd-containing HSV DNA with either 302 nm or 254 nm ultraviolet (UV) light introduced strand breakage into the DNA in a dose-dependent manner when analyzed by alkaline sucrose density gradient sedimentation. Irradiation of unsubstituted HSV DNA under similar conditions produced little strand breakage. These observations are in agreement with the proposed mechanism for photochemical generation of strand breakage in 5-halo-2′-deoxyuridine-containing DNA. Irradiation of IdUrd-substituted virions followed by analysis of the isolated DNA indicated less strand breakage than irradiation of isolated IdUrd-substituted DNA under equivalent conditions. The dosage of irradiation required to introduce DNA strand breakage in IdUrd-substituted virions was equivalent to that employed to affect >99% loss of infectious virus activity in both control and IdUrd-containing virions. It is suggested that the relative UV insensitivity of IdUrd-substituted HSV may be due to the microstructure environment of the substituted HSV DNA which may favor recombination of the photochemically formed halogenuracil radical pairs.

Differences in timing of replication and maturation of nascent DNA fragments between satellite and main band DNA during the S phase of the first cell cycle in cultured carrot root explants.

Considerable portions of cells in cultured carrot root disks replicate DNA in a synchronous fashion, with a maximum peak of synthesis at 48h after excision of the disks. At the early stage of this first replication phase, a satellite DNA component with a higher buoyant density is preferentially synthesized. DNA from the root explants, pulse-labeled with 3H-thymidine at the early (15h) and middle (48h) synthetic stages, sedimented in the 5-9S region in an alkaline sucrose density gradient. After prolonged labeling or chase incubation, the 3H-radioactivity shifted from the 5-9S components to longer intermediate molecules of 10-11S, 12-13S, 18-20S, and 24-26S with the bulk DNA synthesized at the middle stage, but this shift appeared to be delayed with the satellite DNA synthesized at the early stage. The results of chromatographic fractionation of EcoRI-digests of 2h-labeled DNA, and the EcoRI-digests treated additionally with S1-nuclease over benzoylated naphthoylated DEAE-cellulose, indicated that the relative ratio of DNA molecules containing substantial single-stranded regions to predominantly double-stranded molecules was clearly higher in the early replicating DNA.

DNA breakdown of 3T3 and SV 40-transformed 3T3 cells cultured in suspension

It was examined what effect of suspension culture exerted on prelabeled DNA of 3T3 and SV 40 transformed cells (SV3T3). On an alkaline sucrose density gradient the small size DNA of 3T3 cells increased with time of suspension, while that of SV3T3 did not. Furthermore, it was demonstrated that prelabeled DNA of suspended 3T3 cells became small on a neutral sucrose density gradient, in an alkaline and a neutral elution. When SV3T3 cells were treated with dimethylsulfoxide, the smaller DNA appeared on an alkaline sucrose density gradient.

Use of in vivo/in vitro unscheduled DNA synthesis for identification of organ-specific carcinogens.

There are still only a few in vivo short-term assay methods for predicting potential organ-specific carcinogens and mutagens in mammals, although such methods are required for evaluating the in vivo effects of in vitro mutagens. In the in vivo/in vitro UDS assay methods described here, chemicals are given to experimental animals and induction of UDS in target organs is determined by in vitro organ culture or primary cell culture in the presence of [3H]dThd. Incorporation of [3H]dThd into DNA is measured with a liquid scintillation counter or by autoradiography. These methods have now been applied to the glandular stomach, forestomach, colon, liver, kidney, pancreas, tracheal epithelium, nasal epithelium, and spermatocytes. With minor modifications, they may also be applied to other organs. The present review shows that induction of UDS in various organs correlated well with the induction of cancer in these organs. The present authors have used the present methods to identify some potential organ-specific mutagens and carcinogens in mammals. The present authors found that three dicarbonyl compounds, glyoxal, methylglyoxal, and diacetyl, induced apparent UDS and TDS in the glandular stomach, and other groups found that 2-NT, MA6BT, and CNEt6BT induced UDS in the liver. These in vivo/in vitro UDS assays are better than in vitro UDS assay for identification of potential organ-specific mutagens and carcinogens in mammals and are especially useful for identifying potential mutagens and carcinogens that are specific for certain organs, such as the stomach, liver, and kidney. They are also useful for examining the potential mutagenicities and carcinogenicities of carcinogen analogs. However, these methods are not suitable for general in vivo screening because they are not yet available for all organs. A further advantage of the methods is that they can be used to examine larger numbers of animals at one time than other methods for detecting DNA damage, such as alkaline elution or alkaline sucrose density gradient centrifugation. Glyoxal enhanced cancer induction in the glandular stomach by the administration of a limited amount of MNNG and then glyoxal afterward in the two-stage stomach carcinogenesis.

Increased ionic strength: effects on DNA fragmentation and poly(ADP-ribose) polymerase activity in HeLa nuclei.

Poly(ADP-ribose) polymerase activity was measured in a crude nuclear fraction isolated from HeLa cells. It was found that the addition of ammonium sulfate or other salts to the standard incubation medium inhibited the formation of poly(ADP-ribose). Through the use of alkaline sucrose density gradients it was also noted that this same increase in ionic strength inhibited the in vitro breakdown of the HeLa DNA. Additional experiments with alkaline sucrose density gradients and deoxyribonuclease I showed that the in vitro activity of poly(ADP-ribose) polymerase is largely dependent upon DNA fragmentation but that DNA fragmentation at least in vitro is not dependent upon the formation of poly(ADP-ribose). These observations imply that this nuclear enzyme is not extremely sensitive to changes in the ionic strength of the reaction media but is affected indirectly, supposedly through changes in the endonuclease activity of the HeLa nuclei. If this proves to be true, then the addition of salt to the incubation medium for poly(ADP-ribose) polymerase could prove to be a valuable tool for the study of ADP-ribosylation reactions.

Toxicity and DNA damage induced by 1-nitropyrene and its derivatives in Chinese hamster lung fibroblasts

1-Nitropyrene and its chemically synthesised derivatives were investigated for their cytotoxicity and ability to induce DNA-strand breaks in Chinese hamster lung fibroblasts. Both 1-nitrosopyrene (0.25–60 μg/ml) and 1-aminopyrene (0.25–25 μg/ml) were cytotoxic, and induced the formation of DNA lesions, which were measured as DNA sigle-strand breaks after sedimentation in alkaline sucrose-density gradients. Higher doses of 1-aminopyrene (25–60 μg/ml) inhibited the formation of DNA single-strand breaks. 1-Nitropyrene was not toxic (0.25–60 μg/ml)_and induced low levels of detectable DNA strand breaks, whilst N-acetyl-1-aminopyrene was inactive. The post-mitochondrial supernatant fraction of Aroclor-induced rat-liver containing 4 mM NADPH (S9 mix) did not promote the activation of 1-nitropyrene. In fact DNA strand breaks induced by either 1-nitropyrene or 1-nitrosopyrene was abolished in the presence of S9 mix. The 1-nitropyrene reduced intermediate, N-hydroxy-1-aminopyrene was synthesised by the reduction of 1-nitrosopyrene with ascorbic acid. In the presence of ascorbic acid, 1-nitrosopyrene caused a 5-fold increase in the number of DNA single-strand breaks when compared to cells treated with 1-nitrosopyrene alone. The results are discussed in terms of the metabolic activation of 1-nitropyrenre and 1-aminopyrene in Chinese hamster lung cells.

DNA breakage of human leukemia cells by clomiphene, an ovulation-inducing agent

When HPB-ALL, human lymphoblastic leukemia cells were treated with clomiphene citrate, an ovulation-inducing agent, poly(ADP-ribose) synthesizing activity of the cells increased up to 4 fold. This stimulatory effect was almost comparable to that of bleomycin, a typical DNA strand breaking agent. Since the agents causing DNA breakage stimulate poly(ADP-ribose) synthesis in cells [Berger, N. A., Sikorski, G. W., Petzold, S. J., and Kurohara, K. K. J. Clin. Invest. 63, 1164-1171 (1979)], clomiphene citrate is suggested to damage DNA of the cells. In fact, an increase of single strand breakage of the DNA was detected by using alkaline sucrose density gradient centrifugation when the HPB-ALL cells were treated with increasing concentration of clomiphene. The inhibition of the cell growth by clomiphene citrate (IC50 = 5 micrograms/ml) appeared to be ascribable to its potent DNA-damaging effect. Although bleomycin activated purified poly(ADP-ribose) synthetase by cleaving covalently closed circular plasmid pBR322 DNA in vitro, clomiphene citrate per se did not.

Effects of aphidicolin and/or 2‘,3‘-dideoxythymidine on DNA repair induced in HeLa cells by four types of DNA-damaging agents.

The alkaline sucrose density gradient centrifugation method was modified to permit detection of 1 lesion/10(9) daltons of DNA. With this technique, the involvements of DNA polymerases in DNA repair of damage by dimethyl sulfate, UV irradiation, neocarzinostatin, and bleomycin were studied in HeLa cells with the aid of the DNA polymerase inhibitors aphidicolin and 2',3'-dideoxythymidine. DNA repair after UV-induced damage seemed to involve only polymerase alpha, while repair of damage by the other three agents involved both polymerase alpha and a non-alpha polymerase, probably polymerase beta. But repair after damage by dimethyl sulfate differed from that after damage by neocarzinostatin or bleomycin with respect to the co-operations of polymerase alpha and polymerase beta: in repair of dimethyl sulfate-induced damage, both polymerases operated on the same lesions, whereas after damage by neocarzinostatin or bleomycin, polymerase alpha and polymerase beta functioned independently on different lesions.