UV light-induced cyclobutane pyrimidine dimers are mutagenic in mammalian cells.

We used a simian virus 40-based shuttle vector plasmid, pZ189, to determine the role of pyrimidine cyclobutane dimers in UV light-induced mutagenesis in monkey cells. The vector DNA was UV irradiated and then introduced into monkey cells by transfection. After replication, vector DNA was recovered from the cells and tested for mutations in its supF suppressor tRNA marker gene by transformation of Escherichia coli carrying a nonsense mutation in the beta-galactosidase gene. When the irradiated vector was treated with E. coli photolyase prior to transfection, pyrimidine cyclobutane dimers were removed selectively. Removal of approximately 90% of the pyrimidine cyclobutane dimers increased the biological activity of the vector by 75% and reduced its mutation frequency by 80%. Sequence analysis of 72 mutants recovered indicated that there were significantly fewer tandem double-base changes and G X C----A X T transitions (particularly at CC sites) after photoreactivation of the DNA. UV-induced photoproducts remained (although at greatly reduced levels) at all pyr-pyr sites after photoreactivation, but there was a relative increase in photoproducts at CC and TC sites and a relative decrease at TT and CT sites, presumably due to a persistence of (6-4) photoproducts at some CC and TC sites. These observations are consistent with the fact that mutations were found after photoreactivation at many sites at which only cyclobutane dimers would be expected to occur. From these results we conclude that UV-induced pyrimidine cyclobutane dimers are mutagenic in DNA replicated in monkey cells.     

T4 DNA polymerase (3''-5'') exonuclease, an enzyme for the detection and quantitation of stable DNA lesions: the ultraviolet light example.

Ultraviolet light irradiation of DNA results in the formation of two major types of photoproducts, cyclobutane dimers and 6-4' [pyrimidin-2'-one] -pyrimidine photoproducts. The enzyme T4 DNA polymerase possesses a 3' to 5' exonuclease activity and hydrolyzes both single and double stranded DNA in the absence of deoxynucleotide triphosphate substrates. Here we describe the use of T4 DNA polymerase associated exonuclease for the detection and quantitation of UV light-induced damage on both single and double stranded DNA. Hydrolysis of UV-irradiated single or double stranded DNA by the DNA polymerase associated exonuclease is quantitatively blocked by both cyclobutane dimers and (6-4) photoproducts. The enzyme terminates digestion of UV-irradiated DNA at the 3' pyrimidine of both cyclobutane dimers and (6-4) photoproducts. For a given photoproduct site, the induction of cyclobutane dimers was the same for both single and double stranded DNA. A similar relationship was also found for the induction of (6-4) photoproducts. These results suggest that the T4 DNA polymerase proofreading activity alone cannot remove these UV photoproducts present on DNA templates, but instead must function together with enzymes such as the T4 pyrimidine dimer-specific endonuclease in the repair of DNA photoproducts. The T4 DNA polymerase associated exonuclease should be useful for the analysis of a wide variety of bulky, stable DNA adducts.     

Effect of casein kinase II-mediated phosphorylation on the catalytic cycle of topoisomerase II. Regulation of enzyme activity by enhancement of ATP hydrolysis.

The catalytic activity of topoisomerase II is stimulated approximately 2-3-fold following phosphorylation by casein kinase II (Ackerman, P., Glover, C. V. C., and Osheroff, N. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3164-3168). In order to delineate the mechanism by which the activity of the enzyme is enhanced, the effects of casein kinase II-mediated phosphorylation on the individual steps of the catalytic cycle of Drosophila topoisomerase II were characterized. Phosphorylation did not affect reaction steps that preceded hydrolysis of the enzyme's high energy ATP cofactor. This included enzyme-DNA binding, pre-strand passage DNA cleavage/religation, the double-stranded DNA passage event, and post-strand passage DNA cleavage/religation. In contrast, the rate of topoisomerase II-mediated ATP hydrolysis was stimulated 2.7-fold following phosphorylation by casein kinase II. Since ATP hydrolysis is a prerequisite for enzyme turnover, it is concluded that phosphorylation modulates the overall catalytic activity of topoisomerase II by stimulating the enzyme's ATPase activity.     

Replication and mutagenesis of UV-damaged DNA templates in human and monkey cell extracts.

We have used in vitro DNA replication systems from human HeLa cells and monkey CV-1 cells to replicate a UV-damaged simian virus 40-based shuttle vector plasmid, pZ189. We found that replication of the plasmid was inhibited in a UV fluence-dependent manner, but even at UV fluences which caused damage to essentially all of the plasmid molecules some molecules became completely replicated. This replication was accompanied by an increase (up to 15-fold) in the frequency of mutations detected in the supF gene of the plasmid. These mutations were predominantly G:C-->A:T transitions similar to those observed in vivo. Treatment of the UV-irradiated plasmid DNA with Escherichia coli photolyase to reverse pyrimidine cyclobutane dimers (the predominant UV-induced photoproduct) before replication prevented the UV-induced inhibition of replication and reduced the frequency of mutations in supF to background levels. Therefore, the presence of pyrimidine cyclobutane dimers in the plasmid template appears to be responsible for both inhibition of replication and mutation induction. Further analysis of the replication of the UV-damaged plasmid revealed that closed circular replication products were sensitive to T4 endonuclease V (a pyrimidine cyclobutane dimer-specific endonuclease) and that this sensitivity was abolished by treatment of the replicated DNA with E. coli photolyase after replication but before T4 endonuclease treatment. These results demonstrate that these closed circular replication products contain pyrimidine cyclobutane dimers. Density labeling experiments revealed that the majority of plasmid DNA synthesized in vitro in the presence of bromodeoxyuridine triphosphate was hybrid density whether or not the plasmid was treated with UV radiation before replication; therefore, replication of UV-damaged templates appears to occur by the normal semiconservative mechanism. All of these data suggest that replication of UV-damaged templates occurs in vitro as it does in vivo and that this replication results in mutation fixation.     

Immunoprecipitation of pyrimidine(6-4)pyrimidone photoproducts and cyclobutane pyrimidine dimers in uv-irradiated DNA

Biological studies suggest that a significant proportion of the cytotoxicity observed in mammalian cells after uv irradiation may be due to damage other than cyclobutane dimers in DNA. Although pyrimidine-pyrimidone (6-4) photoproducts have been implicated as major contributors to cell lethality, their induction has been measured at considerably less than cyclobutane pyrimidine dimers when measured by chromatographic techniques. Because the yield of (6-4) photoproducts may be reduced by their lability to extreme heat and pH, we have advised an alternative, immunological quantification which does not require DNA hydrolysis. Affinity-purified rabbit antisera were used to precipitate low molecular weight 32P-labeled PM2 DNA irradiated with increasing fluences of uv light. DNA of known molecular weight was used to determine rates of induction for antibody-binding sites associated with (6-4) photoproducts and cyclobutane dimers. These rates were calculated to be 0.6 (6-4) photoproducts and 1.2 cyclobutane dimers/10(8) Da/J/m2. At low uv fluences (6-4) photoproducts were induced at one-half the rate of cyclobutane dimers, whereas at higher fluences (6-4) photoproducts predominated.     

Restriction enzyme cleavage of ultraviolet-damaged simian virus 40 and pBR322 DNA

Cleavage of specific DNA sequences by the restriction enzymes EcoRI, HindIII and TaqI was prevented when the DNA was irradiated with ultraviolet light. Most of the effects were attributed to cyclobutane pyrimidine dimers in the recognition sequences; the effectiveness of irradiation was directly proportional to the number of potential dimer sites in the DNA. Combining EcoRI with dimer-specific endonuclease digestion revealed that pyrimidine dimers blocked cleavage within one base-pair on the strand opposite to the dimer but did not block cleavage three to four base-pairs away on the same strand. These are the probable limits for the range of influence of pyrimidine dimers along the DNA, at least for this enzyme. The effect of irradiation on cleavage by TaqI seemed far greater than expected for the cyclobutane dimer yield, possibly because of effects from photoproducts flanking the tetranucleotide recognition sequence and the effect of non-cyclobutane (6-4)pyrimidine photoproducts involving adjacent T and C bases.     

(6-4)Photoproducts are removed from the DNA of UV-irradiated mammalian cells more efficiently than cyclobutane pyrimidine dimers

A polyclonal antiserum raised against UV-irradiated DNA can be used to assay cyclobutane pyrimidine dimers and Pyr(6-4)Pyo photoproducts specifically by changing the nature of the 32P-labelled antigen. Pyr(6-4)Pyo photoproducts were removed faster than cyclobutane dimers in UV-irradiated human, hamster and mouse cells. Xeroderma pigmentosum cells from complementation groups A, C and D were deficient in the repair of both lesions.     

DNA topoisomerases as targets for the anticancer drug TAS-103: DNA interactions and topoisomerase catalytic inhibition

TAS-103 is a novel anticancer drug that kills cells by increasing levels of DNA cleavage mediated by topoisomerase II. While most drugs that stimulate topoisomerase II-mediated DNA scission (i.e., topoisomerase II poisons) also inhibit the catalytic activity of the enzyme, they typically do so only at concentrations above the clinical range. TAS-103 is unusual in that it reportedly inhibits the catalytic activity of both topoisomerase I and II and does so at physiologically relevant concentrations [Utsugi, T., et al. (1997) Jpn. J. Cancer Res. 88, 992-1002]. Without a topoisomerase activity to relieve accumulating torsional stress, the DNA tracking systems that promote the action of TAS-103 as a topoisomerase II poison would be undermined. Therefore, the effects of TAS-103 on the catalytic activity of topoisomerase I and II were characterized. DNA binding and unwinding assays indicate that the drug intercalates into DNA with an apparent dissociation constant of approximately 2.2 microM. Furthermore, DNA strand passage assays with mammalian topoisomerase I indicate that TAS-103 does not inhibit the catalytic activity of the type I enzyme. Rather, the previously reported inhibition of topoisomerase I-catalyzed DNA relaxation results from a drug-induced alteration in the apparent topology of the nucleic acid substrate. TAS-103 does inhibit the catalytic activity of human topoisomerase IIalpha, apparently by blocking the DNA religation reaction of the enzyme. The lack of inhibition of topoisomerase I catalytic activity by TAS-103 explains how the drug is able to function as a topoisomerase II poison in treated cells.     

Inhibition of transient gene expression in Chinese hamster ovary cells by cyclobutane dimers and (6-4) photoproducts in transfected ultraviolet-irradiated plasmid DNA

Using a transient gene expression assay to measure host cell reactivation, the effects of cyclobutane dimer and noncyclobutane dimer uv photoproducts on expression of a reporter gene were examined in normal and repair-deficient Chinese hamster ovary (CHO) cell lines. Ultraviolet damage in plasmid pRSV beta gal DNA, containing the Escherichia coli beta-galactosidase gene, resulted in reduced reporter gene expression in both uv-hypersensitive mutant CHO cell lines UV5 and UV61 relative to wild-type, parental AA8 cells. However, the effects of uv irradiation of transfected plasmid DNA on gene activity were reduced in UV61, a mutant with normal (6-4) photoproduct repair, compared to UV5, which is deficient in (6-4) photoproduct repair; this reduction correlated with the intermediate uv-hypersensitivity of UV61. Selective removal of cyclobutane dimers by in vitro photoreactivation of uv-irradiated plasmid DNA prior to transfection substantially increased reporter gene activity in both uv-hypersensitive mutant cell lines. This increase was significantly greater in UV61 than in UV5, consistent with UV5 being deficient in repair of both (6-4) photoproducts and cyclobutane dimers. These results suggest that unrepaired (6-4) photoproducts in transfected pRSV beta gal plasmid DNA are responsible for a significant fraction of the reduction in transient gene expression observed in recipient uv-hypersensitive CHO cell mutants.     

Inhibition of DNA topoisomerase I by natural and synthetic mono- and dimeric protoberberine alkaloids

A series of natural (i.e., 1-7) and synthetic (i.e., 8-23) protoberberine alkaloids were evaluated for their inhibitory activities towards DNA topoisomerase I. Both the natural, monomeric protoberberine alkaloids and their mono-modified congeners showed only minor activities. In contrast, most of the dimeric protoberberine alkaloids, especially compounds 12-22, were highly active, with a similar cleavage efficiency as camptothecin (CPT), a well-known, potent topoisomerase-I inhibitor. Thus, these dimeric compounds are promising candidates to be further elaborated as anticancer leads. The mechanism of topoisomerase-I inhibition seems to be dependent on drug concentration for the dimeric protoberberines. At low concentration, they exhibit similar characteristics as CPT. At high concentration, this ability is mostly lost, and the dimers inhibit the relaxation activity of topoisomerase I. Thus, we suppose that the active, dimeric protoberberines strongly bind to plasmid DNA at elevated drug concentration. This most likely results in blocking the enzyme's access to plasmid DNA, thus inhibiting its relaxation.     

Escherichia coli DNA photolyase reverses cyclobutane pyrimidine dimers but not pyrimidine-pyrimidone (6-4) photoproducts

The effect of purified Escherichia coli DNA photolyase on the UV light-induced pyrimidine-pyrimidone (6-4) photoproduct and cyclobutane pyrimidine dimer was investigated in vitro using enzyme purified from cells carrying the cloned phr gene (map position, 15.7 min). Photoproducts were examined both as site-specific lesions in end-labeled DNA and as chromatographically identified products in uniformly labeled DNA. E. coli DNA photolyase removed cyclobutane dimers but had no activity on pyrimidine-pyrimidone (6-4) photoproducts. Photoreactivation can therefore be used to separate the biological effects of these two UV light-induced molecular lesions.     

Sequence dependence of Drosophila topoisomerase II in plasmid relaxation and DNA binding

The sequence dependence of Drosophila topoisomerase II supercoil relaxation and binding activities has been examined. The DNA substrates used in binding experiments were two fragments from Drosophila heat shock locus 87A7. One of these DNA fragments includes the coding region for the heat shock protein hsp70, and the other includes the intergenic non-coding region that separates two divergently transcribed copies of the hsp70 gene at the locus. The intergenic region was previously shown to have a much higher density of topoisomerase cleavage sites than the hsp70 coding region. Competition nitrocellulose filter binding assays demonstrate a preferential binding of the intergene fragment, and that binding specificity increases with increasing ionic strength. Dissociation kinetics indicate a greater kinetic stability of topoisomerase II complexes with the intergene DNA fragment. To study topoisomerase II relaxation activity, we used supercoiled plasmids that contained the same fragments from locus 87A7 cloned as inserts. The relative relaxation rates of the two plasmids were determined under several conditions of ionic strength, and when the plasmid substrates were included in separate reactions or when they were mixed in a single reaction. The relaxation properties of these two plasmids can be explained by a coincidence of high-affinity binding sites, strong cleavage sites, and sites used during the catalysis of strand passage events by topoisomerase II. Sequence dependence of topoisomerase II catalytic activity may therefore parallel the sequence dependence of DNA cleavage by this enzyme.     

Purification and characterization of a novel UV lesion-specific DNA glycosylase/AP lyase from Bacillus sphaericus

The purification and characterization of a pyrimidine dimer-specific glycosylase/AP lyase from Bacillus sphaericus (Bsp-pdg) are reported. Bsp-pdg is highly specific for DNA containing the cis-syn cyclobutane pyrimidine dimer, displaying no detectable activity on oligonucleotides with trans-syn I, trans-syn II, (6-4), or Dewar photoproducts. Like other glycosylase/AP lyases that sequentially cleave the N--glycosyl bond of the 5' pyrimidine of a cyclobutane pyrimidine dimer, and the phosphodiester backbone, this enzyme appears to utilize a primary amine as the attacking nucleophile. The formation of a covalent enzyme-DNA imino intermediate is evidenced by the ability to trap this protein-DNA complex by reduction with sodium borohydride. Also consistent with its AP lyase activity, Bsp-pdg was shown to incise an AP site-containing oligonucleotide, yielding beta- and delta-elimination products. N-terminal amino acid sequence analysis of this 26 kDa protein revealed little amino acid homology to any previously reported protein. This is the first report of a glycosylase/AP lyase enzyme from Bacillus sphaericus that is specific for cis-syn pyrimidine dimers.     

Retarded excision of pyrimidine dimers in human unstimulated lymphocytes

Using immuno-labelling of cyclobutane pyrimidine dimers (CPDs) in nuclei of peripheral lymphocytes after their UVC-irradiation and cultivation, we have found that within the first four hours of cultivation the CPD-specific fluorescent signal from cell nuclei increased. Earlier, a similar increase in binding of antibody specific for pyrimidine (6-4) pyrimidone photoproducts to undenatured DNA isolated from UV-irradiated Chinese hamster ovary cells was reported (Mitchell et al., 1986). Our experiments showed that nucleotide excision repair enzyme might induce such of DNA modification in lymphocyte nuclei that increased specific antibody binding to DNA fragments with lesions. We suggest that enzymatic formation of open structures in DNA predominated qualitatively over dual-incision and excision of these fragments, and resulted in the enhanced exposure of the pyrimidine dimers in nuclei to specific antibodies. The results evidence that nucleotid excision repair in unstimualted human lymphocytes being deficient in dual incision and removal of UV-induced DNA lesions appear to be capable of performing chromatin relaxation and pre-incision uncoiling of DNA fragments with lesions.     

Flavonoids in Helichrysum pamphylicum inhibit mammalian type I DNA topoisomerase

DNA topoisomerases are important targets for cancer chemotherapy. We investigated the effects of a methanolic extract of Helichrysum pamphylicum on mammalian DNA topoisomerase I via in vitro plasmid supercoil relaxation assays. The extracts manifested a considerable inhibition of the enzyme's activity in a dose-dependent manner. We also performed a HPLC analysis to identify the flavonoid content of the H. pamphylicum extract and tested the identified flavonoids; luteolin, luteolin-4-glucoside, naringenin, helichrysinA and isoquercitrin, on DNA topoisomerase I activity. The measurement of the total antioxidant capacity of the flavonoid standards suggested that the topoisomerase inhibition might be correlated with the antioxidant capacity of the plant.     

Inhibition of semiconservative DNA synthesis in ICR 2A frog cells by pyrimidine dimers and nondimer photoproducts induced by ultraviolet radiation

DNA synthesis was examined in ultraviolet (uv)-irradiated ICR 2A frog cells in which either pyrimidine dimers or nondimer photoproducts represented the major class of DNA lesions. Dimers were induced by exposure of cells to 254 nm uv, while nondimer photoproducts were induced by irradiation of cells with uv produced by a fluorescent sunlamp (FSL) that was filtered through 48A Mylar (removes wavelengths less than 310 nm). The FSL-irradiated cultures were also treated with photoreactivating light (PRL) which removed most of the small number of dimers induced by the irradiation, leaving a relatively pure population of nondimer photoproducts. In addition, cells were exposed to 60Co gamma rays. The cultures were pulse-labeled and the size distribution of the DNA synthesized was estimated using both sucrose gradient sedimentation and alkaline step elution. Using either of these techniques, it was found that the presence of dimers resulted in a reduction principally in the synthesis of high molecular weight (MW) DNA. In contrast, nondimer photoproducts caused a strong inhibition in the synthesis of low MW DNA, as was also observed in gamma-irradiated cells. Hence the induction of pyrimidine dimers in DNA mainly affected the elongation of replicons, whereas nondimer lesions primarily caused an inhibition of replicon initiation.     

Mechanism of action of ferrocene derivatives on the catalytic activity of topoisomerase IIalpha and beta--distinct mode of action of two derivatives

Topoisomerase II is found to be present in two isoforms alpha and beta, and both the isoforms are regulated in cancerous tissue. Development of isoform-specific topoisomerase II poisons has been of great interest for cancer-specific drug targeting. In the present investigation using quantitative structure-activity analysis of ferrocene derivatives, we show that two derivatives of ferrocene, azalactone ferrocene and thiomorpholide amido methyl ferrocene, can preferentially inhibit topoisomerase IIbeta activity. Thiomorpholide amido methyl ferrocene shows higher inhibition of catalytic activity (IC(50) = 50 microM) against topoisomerase IIbeta compared to azalactone ferrocene (IC(50) = 100 microM). The analysis of protein DNA intermediates formed in the presence of these two compounds suggests that azalactone ferrocene readily induces formation of cleavable complex in a dose-dependent manner, in comparison with thiomorpholide amido methyl ferrocene. Both the compounds show significant inhibition of DNA-dependent ATPase activity of enzyme. These results suggest that azalactone ferrocene inhibits DNA passage activity of enzyme leading to the formation of cleavable complex, while thiomorpholide amido methyl ferrocene competes with ATP binding resulting in the inhibition of catalytic activity of enzyme. In summary, thiomorpholide amido methyl ferrocene and azalactone ferrocene show distinctly different mechanisms in inhibition of catalytic activity of topoisomerase IIbeta.