Mutagenesis by site-specific arylamine adducts in plasmid DNA: enhancing replication of the adducted strand alters mutation frequency

Site specifically modified plasmids were used to determine the mutagenic effects of single arylamine adducts in bacterial cells. A synthetic heptadecamer bearing a single N-(guanin-8-yl)-2-aminofluorene (AF) or N-(guanin-8-yl)-2-(acetylamino)fluorene (AAF) adduct was used to introduce the adducts into a specific site in plasmid DNA that contained a 17-base single-stranded region complementary to the modified oligonucleotide. Following transformation of bacterial cells with the adduct-bearing DNA, putative mutants were detected by colony hybridization techniques that allowed unbiased detection of all mutations at or near the site of the adduct. The site-specific AF or AAF adducts were also placed into plasmid DNA that contained uracil residues on the strand opposite that bearing the lesions. The presence of uracil in one strand of the DNA decreases the ability of the bacterial replication system to use the uracil-containing strand, thereby favoring the use of the strand bearing the adducts. In a comparison of the results obtained with site specifically modified DNA, either with or without uracil, the presence of the uracil increased the mutation frequencies of the AF adduct by greater than 7-fold to 2.9% and of the AAF adduct by greater than 12-fold to 0.75%. The mutation frequency of the AF adduct was greatly reduced in a uvrA- strain while no mutations occurred with the AAF adduct in this strain. The sequence changes resulting from these treatments were dependent on adduct structure and the presence or absence of uracil on the strand opposite the adducts.(ABSTRACT TRUNCATED AT 250 WORDS)     

A polymerization-based method to construct a plasmid containing clustered DNA damage and a mismatch

Exposure of biological materials to ionizing radiation often induces clustered DNA damage. The mutagenicity of clustered DNA damage can be analyzed with plasmids carrying a clustered DNA damage site, in which the strand bias of a replicating plasmid (i.e., the degree to which each of the two strands of the plasmid are used as the template for replication of the plasmid) can help to clarify how clustered DNA damage enhances the mutagenic potential of comprising lesions. Placement of a mismatch near a clustered DNA damage site can help to determine the strand bias, but present plasmid-based methods do not allow insertion of a mismatch at a given site in the plasmid. Here, we describe a polymerization-based method for constructing a plasmid containing clustered DNA lesions and a mismatch. The presence of a DNA lesion and a mismatch in the plasmid was verified by enzymatic treatment and by determining the relative abundance of the progeny plasmids derived from each of the two strands of the plasmid.     

Isolation and characterization of enhanced fluorescence mutants of Rhodopseudomonas capsulata.

After enrichment by a tetracycline suicide under conditions nonpermissive for the growth of mutants defective in photosynthesis, colonies were screened for enhanced fluorescence in near-infrared light by using high-speed infrared photography. Twenty mutants were isolated, and the chromatophore membranes were analyzed by a new, rapid microprocedure that revealed many different phenotypes among the mutants. The enhanced fluorescence mutants typically possessed a functional light-harvesting II antenna, but showed reduced or absent light-harvesting I. Twelve isolates were also defective in reaction center polypeptides. An R-prime plasmid that bears 50 kilobases of Rhodopseudomonas capsulata DNA coding for components of the photosynthetic apparatus (B. L. Marrs, J. Bacteriol. 146:1003-1012, 1981), pRPS404, complemented all 20 enhanced fluorescence mutants as demonstrated by the quenching of fluorescence in mutants that had received the R-prime plasmid by conjugation. Fluorescence was regained upon loss of the 50-kilobase insert. Complementation of the fluorescent lesions implies that most or all of the genes necessary for the expression of the reaction center and the light-harvesting antennae are carried by the R-prime plasmid and that these genes are actively transcribed in the homologous organism. All 20 mutants are complemented by one of two pBR322 subclones of the R-prime plasmid, pRPSEB2 or pRPSE2. pRPSEB2 bears a 4.5-kilobase fragment of R. capsulata DNA including the rxcA locus, and pRPSE2 is a pBR322 derivative bearing a 7.5-kilobase R. capsulata DNA fragment bearing the rxcB locus. These fragments therefore carry sequences necessary for the normal synthesis of the light-harvesting and reaction center polypeptide complexes.     

Transduction of plasmid determinants in Staphylococcus aureus and Escherichia coli.

Buoyant density analysis of transducing lysates derived from Staphylococcus aureus and Escherichia coli indicated that phage particles bearing plasmid determinants contain a quantity of DNA equivalent to that found in the lytic particles. Transducing particles that bear plasmid determinants smaller than viral DNA must therefore contain a quantity of DNA in excess of a single plasmid genome. In the E. coli P1vir system, a dependence upon host-mediated recombination for the transduction of small plasmids, but not for large R factors or chromosomal genes, was observed. However, no evidence for the involvement of such functions in the transduction of S. aureus plasmids was obtained. Although the origin of the additional DNA in plasmid transducing particles has not been identified, circumstantial evidence has been presented in the staphylococcal system indicating that transducing particles carrying a small tetracycline plasmid are not formed by the wrapping of multiple copies of this plasmid DNA.     

Incision of trivalent chromium [Cr(III)]-induced DNA damage by Bacillus caldotenax UvrABC endonuclease

Some hexavalent chromium [Cr(VI)]-containing compounds are lung carcinogens. Once within cells, Cr(VI) is reduced to trivalent chromium [Cr(III)] which displays an affinity for both DNA bases and the phosphate backbone. A diverse array of genetic lesions is produced by Cr including Cr-DNA monoadducts, DNA interstrand crosslinks (ICLs), DNA-Cr-protein crosslinks (DPCs), abasic sites, DNA strand breaks and oxidized bases. Despite the large amount of information available on the genotoxicity of Cr, little is known regarding the molecular mechanisms involved in the removal of these lesions from damaged DNA. Recent work indicates that nucleotide excision repair (NER) is involved in the processing of Cr-DNA adducts in human and rodent cells. In order to better understand this process at the molecular level and begin to identify the Cr-DNA adducts processed by NER, the incision of CrCl(3) [Cr(III)]-damaged plasmid DNA was studied using a thermal-resistant UvrABC NER endonuclease from Bacillus caldotenax (Bca). Treatment of plasmid DNA with Cr(III) (as CrCl(3)) increased DNA binding as a function of dose. For example, at a Cr(III) concentration of 1 microM we observed approximately 2 Cr(III)-DNA adducts per plasmid. At this same concentration of Cr(III) we found that approximately 17% of the plasmid DNA contained ICLs ( approximately 0.2 ICLs/plasmid). When plasmid DNA treated with Cr(III) (1 microM) was incubated with Bca UvrABC we observed approximately 0.8 incisions/plasmid. The formation of endonuclease IV-sensitive abasic lesions or Fpg-sensitive oxidized DNA bases was not detected suggesting that the incision of Cr(III)-damaged plasmid DNA by UvrABC was not related to the generation of oxidized DNA damage. Taken together, our data suggest that a sub-fraction of Cr(III)-DNA adducts is recognized and processed by the prokaryotic NER machinery and that ICLs are not necessarily the sole lesions generated by Cr(III) that are substrates for NER.     

Sequence-specific p53 gene damage by chloroacetaldehyde and its repair kinetics in Escherichia coli

Oxidative stress and certain environmental carcinogens, e.g. vinyl chloride and its metabolite chloroacetaldehyde (CAA), introduce promutagenic exocyclic adducts into DNA, among them 1,N(6)-ethenoadenine (epsilonA), 3,N(4)-ethenocytosine (epsilonC) and N(2),3-ethenoguanine (epsilonG). We studied sequence-specific interaction of the vinyl-chloride metabolite CAA with human p53 gene exons 5-8, using DNA Polymerase Fingerprint Analysis (DPFA), and identified sites of the highest sensitivity. CAA-induced DNA damage was more extensive in p53 regions which revealed secondary structure perturbations, and were localized in regions of mutation hot-spots. These perturbations inhibited DNA synthesis on undamaged template. We also studied the repair kinetics of CAA-induced DNA lesions in E. coli at nucleotide resolution level. A plasmid bearing full length cDNA of human p53 gene was modified in vitro with 360 mM CAA and transformed into E. coli DH5alpha strain, in which the adaptive response system had been induced by MMS treatment before the cells were made competent. Following transformation, plasmids were re-isolated from transformed cultures 35, 40, 50 min and 1-24 h after transformation, and further subjected to LM-PCR, using ANPG, MUG and Fpg glycosylases to identify the sites of DNA damage. In adaptive response-induced E. coli cells the majority of DNA lesions recognized by ANPG glycosylase were removed from plasmid DNA within 35 min, while MUG glycosylase excised base modifications only within 50 min, both in a sequence-dependent manner. In non-adapted cells resolution of plasmid topological forms was perturbed, suggesting inhibition of one or more bacterial topoisomerases by unrepaired epsilon-adducts. We also observed delayed consequences of DNA modification with CAA, manifesting as secondary DNA breaks, which appeared 3 h after transformation of damaged DNA into E. coli, and were repaired after 24 h.     

Comparative study of the lethal effects of near-UV light (360 nm) and 8-methoxypsoralen plus near-UV on plasmid DNA

We have studied the lethality produced on pBR322 by near-UV radiation and by 8-Methoxypsoralen plus near-UV (PUV treatment). Samples of pBR322 DNA were irradiated with increasing fluences of 360 nm-light either in the absence or presence of 400 molecules of 8-Methoxypsoralen (8-MOP) per plasmid molecule. We have estimated to what extent the global lethality of PUVA treatment is due to the presence of psoralen adducts in DNA or to radiation itself. In order to analyse the involvement of DNA repair mechanisms in the removal of plasmid lesions, several strains of E. coli (differing in their repair capacities) were used as recipients of the treated plasmids. Results showed that excision and recombination participate in the repair of near-UV-induced plasmid lesions. Repair of PUV-induced lesions showed an even greater requirement of the excision pathway. Besides, a slight increase on plasmid mutation frequencies was observed after near-UV or PUV treatment in wild type and uvrA cells. Estimation of the contribution of 8-MOP to the global lethality of PUV treatment showed that only the excision pathway was involved in removing psoralen adducts from plasmid DNA, suggesting the involvement of the recombinational pathway in the repair of near-UV-derived lesions.     

Pathways of Transformation in Ustilago Maydis Determined by DNA Conformation

Ustilago maydis was transformed by plasmids bearing a cloned, selectable gene but lacking an autonomously replicating sequence. Transformation was primarily through integration at nonhomologous loci when the plasmid DNA was circular. When the DNA was made linear by cleavage within the cloned gene, the spectrum of integration events shifted from random to targeted recombination at the resident chromosomal allele. In a large fraction of the transformants obtained using linear DNA, the plasmid DNA was not integrated but was maintained in an extrachromosomal state composed of a concatameric array of plasmid units joined end-to-end. The results suggest the operation of several pathways for transformation in U. maydis, and that DNA conformation at the time of transformation governs choice of pathways.     

Transfer region of IncI1 plasmid R64 and role of shufflon in R64 transfer.

To locate the transfer region of the 122-kiloase plasmid R64drd-11 belonging to incompatibility group I1, a series of deletion derivatives was constructed by in vitro recombinant DNA techniques followed by double homologous recombination in vivo. A plasmid designated pKK609 and bearing a 56.7-kilobase R64 sequence was the smallest transferable plasmid. A plasmid designated pKK610 and no longer possessing the 44-base-pair sequence of the R64 transfer system is located at one end. The other end of the R64 transfer region comprises a DNA segment of about 19 kilobases responsible for pilus formation. Shufflon, DNA with a novel rearrangement in R64, was found to be involved in pilus formation.     

Simple and rapid preparation of gapped plasmid DNA for incorporation of oligomers containing specific DNA lesions

The cytotoxicity, mutagenicity, and carcinogenicity of DNA base lesions are largely determined by the responses of cellular DNA repair proteins, DNA polymerases, and signaling pathways. Elucidation of these processes is thus of high biochemical interest. Such studies increasingly rely on DNA substrates containing specific lesions at defined locations. Although short synthetic DNA oligomers have frequently proved useful, circular plasmid substrates are preferable for much biochemical work, and essential for in vivo studies. However, the complexity of current approaches for preparing such substrates and limitations inherent in the procedures have posed problems. We present here a simple, highly versatile procedure for preparing gapped duplex plasmids, into which oligomers incorporating specific lesions can easily be inserted. Endonuclease N.BstNBI was used to nick twice the same strand of a pUC19-derived plasmid (pUC19HBDa), at two GAGTCNNNN sequences separated by 22 bases. Removal of the 22-nt oligomer and further purification produced a highly pure gapped plasmid. To illustrate application of this procedure, 22-nt oligonucleotides containing a single uracil residue were ligated into the gapped molecules. The pUC19HB(Da) plasmid can be modified to accept almost any DNA-lesion-containing oligomer. Using this new approach to incorporate specific DNA lesions into popular reporter genes will facilitate in vivo study of cellular responses to DNA damage.     

Identification of a centromeric activity in the autonomously replicating TRA region allows improvement of the host-vector system for Candida maltosa

A centromeric activity was identified in the previously isolated 3.8 kb DNA fragment that carries an autonomously replicating sequence (ARS) from the yeast Candida maltosa. Plasmids bearing duplicated copies of the centromeric DNA (dicentric plasmids) were physically unstable and structural rearrangements of the dicentric plasmids occurred frequently in the transformed cells. The centromeric DNA activity was dissociated from the ARS, which is 0.2 kb in size, and was delimited to a fragment at least 325 by in length. The centromeric DNA region included the consensus sequences of CDEI (centromeric DNA element I) and an AT-rich CDEII-like region of Saccharomyces cerevisiae but had no homology to the functionally critical CDEIII consensus. A plasmid bearing the whole 3.8 kb fragment was present in 1–2 copies per cell and was maintained stably even under non-selective culture conditions, while a plasmid having only the 0.2 kb ARS was unstable and accumulated to high copy numbers. The high-copy-number plasmid allowed us to overexpress a gene to a high level, which had never been attained before, under the control of both constitutive and inducible promoters in C. maltosa.     

Excision process of integrated plasmid pBD12 from the Bacillus subtilis chromosome

We studied the behavior of pBD12 plasmid integrated into Bacillus subtilis chromosome via homologous recombination. One copy of the plasmid was integrated into the chromosome, it conferred resistance to low concentrations of antibiotics. Clones with enhanced resistance bearing autonomous plasmid DNAs appeared with a frequency 10(-6) in rec+ but not in recE strain with the integrated plasmid. By restriction and hybridization analysis of some excised plasmids, the sites of excision were determined, chromosomal location of pBD12 plasmid was found to be at the terminal fragment of prophage DNA, so that the att site of phi 105 phage is supposed to be situated on the EcoRI fragment of phage DNA.     

Phasmids. Properties and the use in genetic engineering. II. Packing in vitro. Lysogeny of Escherichia coli K-12

Circular monomeric lambda DNA molecules were used as a substrate for packaging reaction in vitro. For obtaining lambda DNA in circular monomeric form only, Escherichia coli recA plasmid bearing cells were used. This hybrid DNA molecule which we designated phasmid lambda pMYF11, is the pBR322 plasmid in which lambda 47.1 DNA was introduced in vitro. The phasmid can exist in the plasmid form or as a non-defective phage. The efficiency of packaging reaction in vitro proved to be similar for monomeric circular and linear form of phasmid DNA molecules. The cI- variant of the phasmid is not able to exist as a plasmid even in the cells containing homoimmune prophage. Still, cI+ phasmid variants capable of lysogenizing arise with low frequency, as a result of recombination between the resident cI+ prophage and infecting cI- phasmid.     

Generation of linear multigenome-length plasmid molecules in Bacillus subtilis.

Linear multigenome-length double and single stranded plasmid DNA was identified in a Bacillus subtilis ATP-dependent DNAase mutant strain (addA5) bearing plasmids pC194 or pBD95ts. Plasmid pBC30, a seg mutant of pC194, as well as some pUB110 derivatives with rearrangements external to the minimal replicon, produce high amounts of such a concatemeric DNA, even in Rec+ cells. The synthesis of this type of plasmid DNA occurs in the absence of an active plasmid-encoded Rep protein and is markedly affected in polA5 and recE4 genetic backgrounds. To account for these observations, we propose that the AddAB complex serves to prevent a sigma-type replication of plasmid DNA.     

Identification of a centromeric activity in the autonomously replicating TRA region allows improvement of the host-vector system for Candida maltosa

A centromeric activity was identified in the previously isolated 3.8 kb DNA fragment that carries an autonomously replicating sequence (ARS) from the yeast Candida maltosa. Plasmids bearing duplicated copies of the centromeric DNA (dicentric plasmids) were physically unstable and structural rearrangements of the dicentric plasmids occurred frequently in the transformed cells. The centromeric DNA activity was dissociated from the ARS, which is 0.2 kb in size, and was delimited to a fragment at least 325 by in length. The centromeric DNA region included the consensus sequences of CDEI (centromeric DNA element I) and an AT-rich CDEII-like region of Saccharomyces cerevisiae but had no homology to the functionally critical CDEIII consensus. A plasmid bearing the whole 3.8 kb fragment was present in 1–2 copies per cell and was maintained stably even under non-selective culture conditions, while a plasmid having only the 0.2 kb ARS was unstable and accumulated to high copy numbers. The high-copy-number plasmid allowed us to overexpress a gene to a high level, which had never been attained before, under the control of both constitutive and inducible promoters in C. maltosa.     

Repair synthesis by human cell extracts in cisplatin-damaged DNA is preferentially determined by minor adducts.

During reaction of cis-diamminedichloroplatinum(II) (cis-DDP) with DNA, a number of adducts are formed which may be discriminated by the excision-repair system. An in vitro excision-repair assay with human cell-free extracts has been used to assess the relative repair extent of monofunctional adducts, intrastrand and interstrand cross-links of cis-DDP on plasmid DNA. Preferential removal of cis-DDP 1,2-intrastrand diadducts occurred in the presence of cyanide ions. In conditions where cyanide treatment removed 85% of total platinum adducts while approximately 70% of interstrand cross-links remained in plasmid DNA, no significant variation in repair synthesis by human cell extracts was observed. Then, we constructed three types of plasmid DNA substrates containing mainly either monoadducts, 1,2-intrastrand cross-links or interstrand cross-links lesions. The three plasmid species were modified in order to obtain the same extent of total platinum DNA adducts per plasmid. No DNA repair synthesis was detected with monofunctional adducts during incubation with human whole cell extracts. However, a two-fold increase in repair synthesis was found when the proportion of interstrand cross-links in plasmid DNA was increased by 2-3 fold. These findings suggest that (i) cis-DDP 1,2-intrastrand diadducts are poorly repaired by human cell extracts in vitro, (ii) among other minor lesions potentially cyanide-resistant, cis-DDP interstrand cross-links represent a major lesion contributing to the repair synthesis signal in the in vitro assay. These results could account for the drug efficiency in vivo.     

Specificity of replicative and SOS-inducible DNA polymerases in frameshift mutagenesis: mutability of Salmonella typhimurium strains overexpressing SOS-inducible DNA polymerases to 30 chemical mutagens

DNA replication is frequently hindered because of the presence of DNA lesions induced by endogenous and exogenous genotoxic agents. To circumvent the replication block, cells are endowed with multiple specialized DNA polymerases that can bypass a variety of DNA damage. To better understand the specificity of specialized DNA polymerases to bypass lesions, we have constructed a set of derivatives of Salmonella typhimurium TA1538 harboring plasmids carrying the polB, dinB or mucAB genes encoding Escherichia coli DNA polymerase II, DNA polymerase IV or DNA polymerase RI, respectively, and examined the mutability to 30 chemicals. The parent strain TA1538 possesses CGCGCGCG hotspot sequence for -2 frameshift. Interestingly, the chemicals could be classified into four groups based on the mutagenicity to the derivatives: group I whose mutagenicity was highest in strain YG5161 harboring plasmid carrying dinB; group II whose mutagenicity was almost equally high in strain YG5161 and strain TA98 harboring plasmid carrying mucAB; group III whose mutagenicity was highest in strain TA98; group IV whose mutagenicity was not affected by the introduction of any of the plasmids. Introduction of plasmid carrying polB did not enhance the mutagenicity except for benz[a]anthracene. We also introduced a plasmid carrying polA encoding E. coli DNA polymerase I to strain TA1538. Strikingly, the introduction of the plasmid reduced the mutagenicity of chemicals belonging to groups I, II and III, but not the chemicals of group IV, to the levels observed in the derivative whose SOS-inducible DNA polymerases were all deleted. These results suggest that (i) DNA polymerase IV and DNA polymerase RI possess distinct but partly overlapping specificity to bypass lesions leading to -2 frameshift, (ii) the replicative DNA polymerase, i.e., DNA polymerase III, participates in the mutagenesis and (iii) the enhanced expression of E. coli polA may suppress the access of Y-family DNA polymerases to the replication complex.     

Simultaneous synthesis of human-, mouse- and chimeric epidermal growth factor genes via ''hybrid gene synthesis'' approach.

Simultaneous synthesis of two DNA duplexes encoding human and mouse epidermal growth factors (EGF) was accomplished in a single step. A 174 b.p. DNA heteroduplex, with 16 single and double base pair mismatches, was designed. One strand encoded the human EGF, and the opposite strand indirectly encoded the mouse EGF. The heteroduplex DNA was synthesized by ligation of seven overlapping oligodeoxyribonucleotides with a linearized plasmid. After transformation in E. coli HB101 (recA 13), the resulting heteroduplex plasmid served as the template in plasmid replication. Two different plasmid progenies bearing either the human or mouse EGF-coding sequence were identified by colony hybridization using the appropriate probes. However, in E. coli JM103, the same process yielded plasmid progenies encoding different chimeric EGF molecules, presumably due to crossover of human and mouse EGF gene sequences.     

Cluster of point mutations predetermined by a quasipalindromic nucleotide sequence in plasmid pBR322 DNA

Development of a cluster of point mutations due to the correction of an imperfect hairpin in plasmid DNA was investigated. Plasmid pBR322 DNA containing opposite double-strand DNA lesions in the region of a quasipalindrome was constructed. For this aim plasmid DNA was cleaved at the BamHI site, and cytosine residues of the sticky ends were modified by O-methylhydroxylamine. Modified linearized plasmid DNA was ligated and used for transformation of E.coli cells. Tetracycline-sensitive transformants were selected, and the mutants were characterized by restriction and sequencing analysis. One mutant contained a cluster of point mutations. The distribution of mutations was consistent with the cluster having arisen through correction of the imperfect hairpin formed by the quasipalindrome.