We describe an assay for determining the frequency and specificity of mutations occurring at hot spots within a population of DNA molecules. The procedure consists of (a) annealing the DNA population with a labeled oligonucleotide designed to prime DNA synthesis at the mutational hot spot; (b) DNA elongation in the presence of a single dideoxynucleoside triphosphate together with 1-3 deoxynucleoside triphosphates, and (c) quantitation of all limit elongation products by high-resolution gel electrophoresis followed by autoradiography and computing densitometry. Derivation of mutational frequency and specificity over a wide range of values is demonstrated for M13 viral DNA mixtures containing defined proportions of wild-type and mutant DNAs, as well as for M13 viral DNA populations obtained by transfection of DNA bearing a defined site-specific ethenocytosine lesion. The assay is shown to yield results similar to those obtained by laborious clone-by-clone sequencing of viral progeny. The method is not affected significantly by several tested variables and appears to be suitable for use as a quantitative assay for sequence microheterogeneity at defined positions within DNA populations. Application of the methodology demonstrates that ethenocytosine, an exocyclic DNA lesion induced by carcinogens such as vinyl chloride and urethane, is a highly efficient mutagenic lesion with a mutational specificity expected for noninstructive lesions.

It is widely accepted that mutagenic DNA lesions fall into two categories: mispairing lesions hydrogen bond with an incorrect incoming base, generally do not stop replication, and possess high mutagenic efficiency without any requirement for induced functions; noninstructional lesions lack accessible template information, act as strong blocks to DNA replication (and are therefore toxic), and their mutagenic effects are SOS-dependent. Our recent results show that ethenocytosine (epsilon C), a noninstructional exocyclic DNA lesion induced by vinyl chloride, may have unusual mutagenic properties. To obtain more definitive experimental evidence for the observed effects, we have introduced a single epsilon C residue at a specific site of coliphage M13AB28 replicative form DNA by a "single-stranded linker-ligation" technique. The resulting DNA was purified and transfected into appropriate recA+ or recA- Escherichia coli host cells. The effect of epsilon C on survival was determined from transfection efficiency. Both the frequency and specificity of mutations induced by epsilon C were determined by direct sequence analysis of randomly picked progeny phage plaques. The results indicated that epsilon C has little effect on the survival of M13 DNA. Approximately 30% of the progeny phage obtained by transfecting epsilon C DNA had a base substitution mutation precisely at the lesion site. No such mutations were observed in progeny plaques obtained by transfecting the control DNA construct. All epsilon C-induced mutations were either C-to-T transitions or C-to-A transversions. Neither survival nor mutagenic efficiency was significantly affected in recA- host cells.(ABSTRACT TRUNCATED AT 250 WORDS)

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The Escherichia coli UVM response is a recently described phenomenon in which pretreatment of cells with DNA-damaging agents such as UV or alkylating agents significantly enhances mutation fixation at a model mutagenic lesion (3,N 4 -ethenocytosine; C) borne on a transfected M13 single-stranded DNA genome. Since UVM is observed in ⌬recA cells in which SOS induction should not occur, UVM may represent a novel, SOS-independent, inducible response. Here, we have addressed two specific hypothetical mechanisms for UVM: (i) UVM results from a recA-independent pathway for the induction of SOS genes thought to play a role in induced mutagenesis, and (ii) UVM results from a polymerase switch in which M13 replication in treated cells is carried out by DNA polymerase I (or DNA polymerase II) instead of DNA polymerase III. To address these hypotheses, E. coli cells with known defects in recA, lexA, umuDC, polA, or polB were treated with UV or 1-methyl-3-nitro-1-nitrosoguanidine before transfection of M13 single-stranded DNA bearing a site-specific ethenocytosine lesion. Survival of the transfected DNA was measured as transfection efficiency, and mutagenesis at the C residue was analyzed by a quantitative multiplex DNA sequencing technology. Our results show that UVM is observable in ⌬recA cells, in lexA3 (noninducible SOS repressor) cells, in LexA-overproducing cells, and in ⌬umuDC cells. Furthermore, our data show that UVM induction occurs in the absence of detectable induction of dinD, an SOS gene. These results make it unlikely that UVM results from a recAindependent alternative induction pathway for SOS genes. Similarly, UVM is observed in polA (deficient in DNA polymerase I) and polB (deficient in DNA polymerase II) cells, suggesting that neither polymerase plays an indispensable role in UVM induction. Furthermore, our data show that the UVM response is accompanied by enhanced survival (UVM reactivation) of M13 DNA bearing C. The observation of UVM reactivation makes simple repair-suppression models for UVM less attractive and increases the plausibility of mechanisms operating at the level of base insertion. We hypothesize that noncoding lesions fall into two categories. The so-called SOS-dependent (class 1) lesions require SOS functions at the extension (bypass) step, whereas class 2 noncoding lesions do not. It is proposed that UVM, a previously unrecognized damage-inducible response, modulates base insertion at noncoding lesions.Efficient replication past certain DNA lesions depends on factors not required for the replication of normal template DNA. In Escherichia coli, the required additional factors are thought to be encoded by the member genes of the SOS regulon. According to the widely accepted SOS hypothesis, unrepaired DNA damage induces the SOS genes, ultimately resulting in enhanced levels of specific gene products that are proposed to transiently alter the replication machinery.The major features of the regulation of the SOS regulon are well described (20,45,50,65,66,69). Under normal conditions, transcript...