The dynamic distribution and quantification of DNA cruciforms in eukaryotic nuclei

Cruciforms have been suggested as potential recognition structures at or near origins of DNA replication in eukaryotic cells. Monoclonal antibodies specific for cruciforms have been produced. The antibody binds to structural determinants at the base of the cruciform stem, the "elbow." Labeling of nuclei with anti-cruciform antibodies produces a nonuniform pattern of fluorescence in cells arrested at the G1/S boundary. This pattern of fluorescence changes when these cells are released from synchrony. Using fluorescence flow cytometry to quantify the number of DNA cruciform structures in cells throughout the cell cycle, we observed two major populations of nuclei with different numbers of cruciforms; the modal number of cruciforms in these populations was 0.6 x 10(5) and 3 x 10(5) cruciforms per nucleus. Synchronized cells (doubly arrested by serum starvation and aphidicolin) displayed a biphasic distribution of the number of cruciforms over the first 6 h after release from synchrony with maxima at 0 and 4 h after release.     

Inverted repeats,stem-loops,and cruciforms: Significance for initiation of DNA replication

Inverted repeats occur nonrandomly in the DNA of most organisms. Stem-loops and cruciforms can form from inverted repeats. Such structures have been detected in pro- and eukaryotes. They may affect the supercoiling degree of the DNA, the positioning of nucleosomes, the formation of other secondary structures of DNA, or directly interact with proteins. Inverted repeats, stem-loops, and cruciforms are present at the replication origins of phage, plasmids, mitochondria, eukaryotic viruses, and mammalian cells. Experiments with anti-cruciform antibodies suggest that formation and stabilization of cruciforms at particular mammalian origins may be associated with initiation of DNA replication. Many proteins have been shown to interact with cruciforms, recognizing features like DNA crossovers, four-way junctions, and curved/bent DNA of specific angles. A human cruciform binding protein (CBP) displays a novel type of interaction with cruciforms and may be linked to initiation of DNA replication. © 1996 Wiley-Liss, Inc.     

Calmodulin-specific monoclonal antibodies inhibit DNA replication in mammalian cells.

The involvement of calmodulin in the proliferation of Chinese hamster embryo fibroblast cells has been studied with a specific monoclonal antibody to calmodulin. We observed that calmodulin levels increase 2-fold in the late G1 period in these cells, and this coincides with the increase in DNA polymerase alpha activity as the cells progress synchronously from a quiescent state in the G1 to the S phase. However, there is a concurrent 10-fold enhancement of thymidine kinase activity, which is tightly coupled to the entry of cells into the S phase. Incubation of permeabilized S-phase cells with calmodulin-specific murine monoclonal antibody resulted in a dose-dependent inhibition of DNA replication. This inhibitory effect of anti-calmodulin antibodies on DNA replication is completely reversed by the addition of exogenously purified calmodulin. These observations provide evidence for the involvement of calmodulin in DNA replication and, therefore, in cell proliferation during the S phase.     

Binding of monoclonal antibody to the Epstein Barr virus (EBV)/CR2 receptor induces activation and differentiation of human B lymphocytes

A panel of B cell-specific monoclonal antibodies that identify the CR2/EBV receptor were examined for their ability to mimic the T-independent mitogenic agent, EBV, and thus activate human peripheral blood B lymphocytes. Two of four different anti-CR2/EBV monoclonal antibodies, OKB7 and AB-1, produced a 50-fold to 200-fold dose-dependent stimulation of DNA synthesis of peripheral blood mononuclear cells. One of the other monoclonal antibodies, anti-B2, had slight activity, and the other, HB-5, was completely inactive. One of the mitogenic antibodies, OKB7, which directly inhibits binding and infection of B cells by EBV in the absence of a second anti-immunoglobulin antibody, was examined in further detail. Both the intact antibody in soluble form and its pepsin-derived F(ab')2 fragment stimulated DNA synthesis of unseparated B and T lymphocytes. Peak stimulation of DNA synthesis in peripheral blood mononuclear cells occurred between 4 to 6 days. B cells were responsible for incorporation of [3H]thymidine. However, T cells were required for activation of peripheral blood mononuclear cells by OKB7. OKB7, as well as the other mitogenic monoclonal anti-EBV/CR2 receptor antibody, also induced B cells to differentiate after 6 to 10 days of culture as indicated by polyclonal Ig secretion. IgM was the predominate immunoglobulin secreted. These studies thus indicate that certain epitopes on the EBV/CR2 receptor trigger B cells to divide and differentiate. This pathway of B cell activation, in contrast to that produced by EBV, is T cell dependent.     

A microinjected monoclonal antibody against human DNA polymerase-alpha inhibits DNA replication in human, hamster, and mouse cell lines

We have examined the effect that microinjection of a monoclonal antibody directed against human DNA polymerase-alpha (SJK-287) has on DNA synthesis in exponentially growing human, mouse, and hamster cell lines. We show that the SJK-287 antibody, when microinjected directly into the nuclei of cells is capable of inhibiting DNA synthesis in all three cell lines tested. Moreover, the effectiveness with which this antibody can inhibit ongoing DNA synthesis by the microinjection assay is closely correlated with the ability of the antibody to neutralize DNA polymerase-alpha activity fractionated from each cell line in vitro. Two other monoclonal antibodies of the same class, one directed against the cellular p53 protein (PAb122), and one directed against the c-myc protein (PM-8) were also tested for their ability to inhibit ongoing DNA synthesis by direct microinjection and in lysolecithin permeabilized cells. Both monoclonal antibodies failed to inhibit ongoing DNA synthesis in exponentially growing cells by these assays.     

DNA repair synthesis in human fibroblasts requires DNA polymerase delta

When UV-irradiated cultured diploid human fibroblasts were permeabilized with Brij-58 then separated from soluble material by centrifugation, conservative DNA repair synthesis could be restored by a soluble factor obtained from the supernatant of similarly treated HeLa cells. Extensive purification of this factor yielded a 10.2 S, 220,000-dalton polypeptide with the DNA polymerase and 3'- to 5'-exonuclease activities reported for DNA polymerase delta II (Crute, J. J., Wahl, A. F., and Bambara, R. A. (1986) Biochemistry 25, 26-36). Monoclonal antibody to KB cell DNA polymerase alpha, while binding to HeLa DNA polymerase alpha, did not bind to the HeLa DNA polymerase delta. Moreover, at micromolar concentrations N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP) and 2-(p-n-butylanilino)-2'-deoxyadenosine 5'-triphosphate (BuAdATP) were potent inhibitors of DNA polymerase alpha, but did not inhibit the DNA polymerase delta. Neither purified DNA polymerase alpha nor beta could promote repair DNA synthesis in the permeabilized cells. Furthermore, under conditions which inhibited purified DNA polymerase alpha by greater than 90%, neither monoclonal antibodies to DNA polymerase alpha, BuPdGTP, nor BuAdATP was able to inhibit significantly the DNA repair synthesis mediated by the DNA polymerase delta. Thus, it appears that a major portion of DNA repair synthesis induced by UV irradiation might be catalyzed by DNA polymerase delta. When xeroderma pigmentosum human diploid fibroblasts were utilized, DNA repair synthesis dependent upon ultraviolet light could be restored by addition of both T4 endonuclease V and DNA polymerase delta, but not by addition of either one alone. This result suggests that cytosol-depleted permeabilized DNA repair-defective human fibroblasts and HeLa DNA polymerase delta might be exploited to provide a functional assay for purifying active DNA repair factors from DNA repair-proficient cells without a preknowledge of their function.     

Identification of DNA polymerase delta in CV-1 cells: studies implicating both DNA polymerase delta and DNA polymerase alpha in DNA replication

DNA polymerases delta and alpha were purified from CV-1 cells, and their sensitivities to the inhibitors aphidicolin, (p-n-butylphenyl)deoxyguanosine triphosphate (BuPdGTP), and monoclonal antibodies directed against DNA polymerase alpha were determined. The effects of these inhibitors on DNA replication in permeabilized CV-1 cells were studied to investigate the potential roles of polymerases delta and alpha in DNA replication. Aphidicolin was shown to be a more potent inhibitor of DNA replication than of DNA polymerase alpha or delta activity. Inhibition of DNA replication by various concentrations of BuPdGTP was intermediate between inhibition of purified polymerase alpha or delta activity. Concentrations of BuPdGTP which totally abolished DNA polymerase alpha activity were much less effective in reducing DNA replication, as well as the activity of DNA polymerase delta. Monoclonal antibodies which specifically inhibited polymerase alpha activity reduced, but did not abolish, DNA replication in permeable cells. BuPdGTP, as well as anti-polymerase alpha antibodies, inhibited DNA replication in a nonlinear manner as a function of time. Depending upon the initial or final rates of inhibition of replication by BuPdGTP and anti-alpha antibodies, as little as 50%, or as much as 80%, of the replication activity can be attributed to polymerase alpha. The remaining replication activity (20-50%) is tentatively attributed to polymerase delta, because it was aphidicolin sensitive and resistant to both anti-polymerase alpha antibodies and low concentrations of BuPdGTP. A concentration of BuPdGTP which abolished polymerase alpha activity reduced, but did not abolish, both the synthesis and maturation of nascent DNA fragments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monoclonal antibodies to cruciform DNA structures

Two monoclonal antibodies, 2D3 and 4B4, have been raised against a cruciform structure in a heteroduplex DNA molecule. Antibody binding to DNA fragments was determined by a radioimmunoassay in which DNA--antibody complexes were separated from unbound DNA by acrylamide gel electrophoresis. These antibodies seem to recognize conformational determinants specific to cruciform structures. 2D3 and 4B4 antibodies do not bind to linear double-stranded homoduplex DNA fragments, linear single-stranded DNA or single-stranded simian virus 40 DNA containing a stem--loop structure, but do bind to the original cruciform and to a different cruciform with one shortened arm. 2D3 also bound to a T-shaped double-stranded DNA molecule, while 4B4 binding to this structure was weak. The monoclonal antibodies 2D3 and 4B4 were found to be immunoglobulin G1 and immunoglobulin M, respectively.     

Effect of DNA polymerase inhibitors on DNA repair in intact and permeable human fibroblasts: evidence that DNA polymerases delta and beta are involved in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine

The involvement of DNA polymerases alpha, beta, and delta in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was investigated in human fibroblasts (HF). The effects of anti-(DNA polymerase alpha) monoclonal antibody, (p-n-butylphenyl)deoxyguanosine triphosphate (BuPdGTP), dideoxythymidine triphosphate (ddTTP), and aphidicolin on MNNG-induced DNA repair synthesis were investigated to dissect the roles of the different DNA polymerases. A subcellular system (permeable cells), in which DNA repair synthesis and DNA replication were differentiated by CsCl gradient centrifugation of BrdUMP density-labeled DNA, was used to examine the effects of the polymerase inhibitors. Another approach investigated the effects of several of these inhibitors on MNNG-induced DNA repair synthesis in intact cells by measuring the amount of [3H]thymidine incorporated into repaired DNA as determined by autoradiography and quantitation with an automated video image analysis system. In permeable cells, MNNG-induced DNA repair synthesis was inhibited 56% by 50 micrograms of aphidicolin/mL, 6% by 10 microM BuPdGTP, 13% by anti-(DNA polymerase alpha) monoclonal antibodies, and 29% by ddTTP. In intact cells, MNNG-induced DNA repair synthesis was inhibited 57% by 50 micrograms of aphidicolin/mL and was not significantly inhibited by microinjecting anti-(DNA polymerase alpha) antibodies into HF nuclei. These results indicate that both DNA polymerases delta and beta are involved in repairing DNA damage caused by MNNG.     

Transfer of monoclonal antibodies into mammalian cells by electroporation

A simple rapid and reproducible procedure for transferring monoclonal antibodies into mammalian cells by electroporation is described. Two functionally different monoclonal antibodies (Mab 3F3 and Mab 2B4) specific for asparagine synthetase (EC 6.3.1.1) were used for electroporation into HeLa, HT-5, and L5178Y D10/R (L-asparaginase-resistant) cells. The conditions were optimized so that the viability of the electroporated cells was very high (80-90%), and 90% of the viable cells had antibody incorporated. Electropermeabilized cells were structurally intact, and the high voltage electric pulse had no inhibitory effect on overall cellular DNA and protein synthesis. Incorporated immunoglobulins showed unaltered structural integrity and were functionally active. L5178Y D10/R cells incorporated with an antibody (Mab 3F3) known to be a potent inhibitor of tumor asparagine synthetase showed increased dependence on an exogenous source of asparagine in the culture medium, while the growth of cells incorporated with a control (noninhibitory) antibody (Mab 2B4) remained unaffected. These studies demonstrate that electroporation can be employed successfully for large scale transfer of antibodies into cultured mammalian cells for the study of cellular metabolism.     

Characterization of the binding specificity of two anticruciform DNA monoclonal antibodies

Two monoclonal antibodies (2D3 and 4B4) have been raised against a stable cruciform DNA structure containing the 27-base pair palindrome of the SV40 origin of replication on one strand and an unrelated 26-base pair palindrome on the complementary strand (pRGM 21 x pRGM 29) and have been shown to recognize conformational determinants specific to cruciform DNA structures (Frappier, L., Price, G.B., Martin, R. G., and Zannis-Hadjopoulos, M. (1987) J. Mol. Biol. 193, 751-758). To define the region(s) of the cruciform that is recognized by these antibodies, we examined the ability of 2D3 and 4B4 to protect the single-stranded tips of the loops or the four-way junctions at the base of the stem of stable cruciform molecules against cleavage by mung bean nuclease or T7 endonuclease 3, respectively. Both antibodies were found to protect two of the four elbow-like structures at the base of the cruciform from T7 endonuclease 3 cleavage, but not the tips of the cruciform arms from mung bean nuclease cleavage. Also, predigestion of the cruciform with mung bean nuclease did not affect the binding of either antibody. In addition, 2D3 bound to a cruciform and a T-shaped structure involving the palindromic sequence at the cloning site of pUC7, which is completely unrelated in sequence to the palindrome of pRGM 21 x pRGM 29, and protected the base of these stem-loop structures against cleavage by T4 endonuclease VII. These results indicate that 2D3 and 4B4 bind at or near the base of the cruciform molecules and that, at least for 2D3, binding is independent of DNA sequence.     

Identification of polypeptide components of the Epstein-Barr virus early antigen complex with monoclonal antibodies.

Three monoclonal antibodies were produced against the Epstein-Barr virus-induced early antigen complex. These antibodies were shown to be specific for the early antigen complex by the fact that they only reacted with cells supporting a permissive or abortive Epstein-Barr virus infection and their synthesis was not affected by inhibitors of viral DNA synthesis. One monoclonal antibody, designated R3, was directed against a diffuse component of the early antigen complex since it reacted by immunofluorescence with cells fixed in acetone or methanol. The other two monoclonal antibodies, designated K8 and K9, reacted with a methanol-sensitive restricted component of this complex. The appearance of the R3 antigen in P3HR-1 superinfected Raji cells occurred approximately 4 h earlier than the antigen detected by K8. By both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and radioimmunoelectrophoresis, it was determined that the R3 monoclonal antibody recognized two major polypeptides with molecular weights of approximately 50,000 to 52,000, whereas K8 and K9 precipitated a protein of approximately 85,000. The R3 monoclonal antibody also immunoprecipitated an in vitro primary translation product. It was, therefore, possible to map this product to the Epstein-Barr virus DNA BamH1 M fragment. These in vitro products were slightly smaller than the in vivo proteins, suggesting that these proteins probably undergo posttranslational modification during the virus replication cycle.     

Suppression of dexamethasone-stimulated DNA synthesis in an oncogene construct containing rat cell line by a DNA site-oriented ligand of poly-ADP-ribose polymerase: 6-amino-1,2-benzopyrone

The cellular inhibitory effects of 6-amino-1,2-benzopyrone (6-ABP), a DNA site-specific ligand of adenosine diphosphoribosyl transferase (ADPRT), were determined in a dexamethasone-sensitive EJ-ras gene construct containing cell line (14C cells). Dexamethasone in vitro transforms these cells to a tumorigenic phenotype and also stimulates cell replication. At a non-toxic concentrations (0.2 mM) 6-ABP treatment of intact cells for 4 days inhibits the dexamethasone-stimulated increment of cellular DNA content, depresses replicative DNA synthesis as assayed by thymidine incorporation to the level of cells that were not exposed to dexamethasone, and in permeabilized cells reduces the dexamethasone-stimulated increase of deoxyribonucleotide incorporation into DNA to the level of untreated cells. In situ pulse labeling of cells pretreated with 6-ABP indicated an inhibition of DNA synthesis at a stage prior to the formation of the 10-kb intermediate species. The drug had no direct effect on cellular DNA polymerases as tested in vitro, and the inhibition of DNA synthesis in permeabilized cells following drug treatment for 4 days was abolished by externally added DNA templates. Neither dexamethasone nor the drug influenced the cellular quantity of ADPRT molecules, tested immunochemically.     

Isolation of the gene encoding yeast DNA polymerase I

A yeast genomic DNA expression library in lambda gt11 antibody prepared against yeast DNA polymerase I were used to isolate the gene encoding DNA polymerase I. The identity of the DNA polymerase I gene was determined by several criteria. First, the clone-encoded protein is immunologically related to DNA polymerase I. Second, cells containing the gene cloned in the high copy number plasmid YEp24 overproduce the polymerase activity 4- to 5-fold as measured in yeast extracts. Finally, insertion of the gene downstream from a bacteriophage T7 promoter allows synthesis of yeast DNA polymerase I in Escherichia coli. Gene disruption and Southern hybridization experiments show that the polymerase is encoded by an essential, single copy gene. Examination of the germinated spores containing the disrupted gene reveals a defect in nuclear division and a terminal phenotype typical of replication mutants.     

A neutralizing antibody against human DNA polymerase epsilon inhibits cellular but not SV40 DNA replication.

The contribution of human DNA polymerase epsilon to nuclear DNA replication was studied. Antibody K18 that specifically inhibits DNA polymerase activity of human DNA polymerase epsilon in vitro significantly inhibits DNA synthesis both when microinjected into nuclei of exponentially growing human fibroblasts and in isolated HeLa cell nuclei. The capability of this neutralizing antibody to inhibit DNA synthesis in cells is comparable to that of monoclonal antibody SJK-132-20 against DNA polymerase alpha. Contrary to the antibody against DNA polymerase alpha, antibody K18 against DNA polymerase epsilon did not inhibit SV40 DNA replication in vitro. These results indicate that DNA polymerase epsilon plays a role in replicative DNA synthesis in proliferating human cells like DNA polymerase alpha, and that this role for DNA polymerase epsilon cannot be modeled by SV40 DNA replication.     

Inhibition of DNA replication and DNA polymerase alpha activity by monoclonal anti-(DNA polymerase alpha) immunoglobulin G and F(ab) fragments

The effect of monoclonal anti-(DNA polymerase alpha) immunoglobulin G (IgG) and F(ab) fragments on DNA replication in lysolecithin-permeabilized human cells and on DNA polymerase alpha activity was determined. DNA polymerase alpha activity in vitro was inhibited equally by the same concentrations of monoclonal IgGs and F(ab) fragments. However, the IgGs and F(ab) fragments were not equally potent in inhibiting DNA replication in permeable cells. In general, the F(ab) fragments were approximately equal to 10-fold more potent than IgGs in inhibiting DNA replication, suggesting the F(ab) fragments cross the nuclear membrane more readily than IgGs. Immunocytochemical studies demonstrated that at least a fraction of anti-(DNA polymerase alpha) IgGs entered the nucleus of permeable cells. For most antibodies tested, the IgG or F(ab) concentration needed to inhibit replication was several orders of magnitude higher than that needed to neutralize polymerase alpha activity extracted from the same number of cells. Anti-(DNA polymerase alpha) F(ab) fragments were shown to inhibit the discontinuous synthesis of Okazaki DNA, as well as the maturation of Okazaki DNA to larger DNA, thereby implicating DNA polymerase alpha in both of these processes.     

Identification of Two Regions in Apolipoprotein B100 that are Exposed on the Cytosolic Side of the Endoplasmic Reticulum Membrane

Protease protection assays of apolipoprotein B100 (apoB) in digitonin-permeabilized HepG2 cells indicated that multiple domains of apoB are exposed to the cytosol through an extensive portion of the secretory pathway. The intracellular orientation of apoB in the secretory pathway was confirmed by immunocytochemistry using antibodies recognizing specific domains of apoB in streptolysin-O (STP-O)– and saponin-permeabilized HepG2 cells. Lumenal epitopes on marker proteins in secretory pathway compartments (p63, p53, and galactosyltransferase) were not stained by antibodies in STP-O–treated cells, but were brightly stained in saponin-treated cells, confirming that internal membranes were not perforated in STP-O–treated cells. An anti-apoB peptide antibody (B4) recognizing amino acids 3221–3240 caused intense staining in close proximity to the nuclear membrane, and less intensely throughout the secretory pathway in STP-O–permeabilized cells. Staining with this antibody was similar in STP-O– and saponin-treated cells, indicating that this epitope in apoB is exposed to the cytosol at the site of apoB synthesis and throughout most of the remaining secretory pathway. Similar results indicating a cytosolic orientation were obtained with monoclonal antibody CC3.4, which recognizes amino acids 690–797 (79–91 kD) in apoB. Two polyclonal antibodies made to human LDL and two monoclonal antibodies recognizing amino acids 1878–2148 (D7.2) and 3214–3506 (B1B6) in apoB did not produce a strong reticular signal for apoB in STP-O–treated cells. The anti-LDL and B1B6 antibodies produced almost identical punctate patterns in STP-O–treated cells that overlapped with LAMP-1, a membrane marker for lysosomes. These observations suggest that the B1B6 epitope of apoB is exposed on the surface of the lysosome. The results identify two specific regions in apoB that are exposed to the cytosol in the secretory pathway.     

Monoclonal antibody specific for chicken DNA polymerase alpha associated with DNA primase

Four monoclonal antibodies against chicken DNA polymerase alpha were obtained from mouse hybridomas (see ref. 1). Two of them, 4-2D and 4-8H, recognized different epitopes of the DNA polymerase alpha-DNA primase complex as determined by a competitive enzyme-linked immunosorbent assay. Antibody 4-8H partially (about 30%) neutralized the combined activity of primase-DNA polymerase alpha as well as the DNA polymerase alpha activity. In contrast, antibody 4-2D did not neutralize DNA polymerase alpha activity, but neutralized the primase-DNA polymerase alpha activity extensively (up to 80%). Furthermore, although an immunoaffinity column made with 4-8H antibody retained virtually all of the DNA polymerase alpha with and without associated primase, a column made with 4-2D antibody did not bind DNA polymerase alpha without the primase, but retained the enzyme associated with the primase. These results indicate that 4-8H monoclonal antibody is specific for DNA polymerase alpha and 4-2D monoclonal antibody is specific for the primase or a special structure present in the primase-DNA polymerase alpha complex.     

The 14-3-3 protein homologues from Saccharomyces cerevisiae,Bmh1p and Bmh2p,have cruciform DNA-binding activity and associate in vivo with ARS307

We have previously shown that, in human cells, cruciform DNA-binding activity is due to 14-3-3 proteins (Todd, A., Cossons, N., Aitken, A., Price, G. B., and Zannis-Hadjopoulos, M. (1998) Biochemistry 37, 14317-14325). Here, wild-type and single- and double-knockout nuclear extracts from the 14-3-3 Saccharomyces cerevisiae homologues Bmh1p and Bmh2p were analyzed for similar cruciform-binding activities in relation to these proteins. The Bmh1p-Bmh2p heterodimer, present in the wild-type strain, bound efficiently to cruciform-containing DNA in a structure-specific manner because cruciform DNA efficiently competed with the formation of the complex, whereas linear DNA did not. In contrast, the band-shift ability of the Bmh1p-Bmh1p and Bmh2p-Bmh2p homodimers present in the bmh2(-) and bmh1(-) single-knockout cells, respectively, was reduced by approximately 93 and 82%, respectively. The 14-3-3 plant homologue GF14 was also able to bind to cruciform DNA, suggesting that cruciform-binding activity is a common feature of the family of 14-3-3 proteins across species. Bmh1p and Bmh2p were found to associate in vivo with the yeast autonomous replication sequence ARS307, as assayed by formaldehyde cross-linking, followed by immunoprecipitation with anti-Bmh1p/Bmh2p antibody and conventional PCR. In agreement with the finding of an association of Bmh1p and Bmh2p with ARS307, another immunoprecipitation experiment using 2D3, an anti-cruciform DNA monoclonal antibody, revealed the presence of cruciform-containing DNA in ARS307.