Changes in DNA base sequence induced by targeted mutagenesis of lambda phage by ultraviolet light

Wood, Richard D.; Skopek, Thomas R.; Hutchinson, Franklin

doi:10.1016/0022-2836(84)90121-9

Cited by 138 publications

(54 citation statements)

References 37 publications

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“…Application of the lad shuttle to study the mutagenic specificity of UV light in human cells has revealed a pattern strikingly similar to that reported for E. coli (6,(14)(15)(16)(17)(18). The majority of the 53 mutations analyzed (81%) are G-C-to-A-T transitions.…”

Section: Methodssupporting

confidence: 71%

The lacI shuttle: rapid analysis of the mutagenic specificity of ultraviolet light in human cells.

Lebkowski¹,

Clancy²,

Miller³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

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A system has been devised that allows the effect of mutagens acting in human cells to be readily analyzed at the DNA sequence level. The bacterial gene WIcI, carried on a shuttle vector, is introduced into human tissue culture cells by transfection and allowed to replicate in the cell nucleus.Twenty-four to 48 hr after transfection, the cells are exposed to a mutagen. After 1-2 days of further replication, vector DNA is purified and transfected back intoEscherichia coli for scoring and analysis of mutations in laW. The (1)(2)(3)(4). In these experiments, the target gene is carried on a shuttle vector, which can replicate in both mammalian and bacterial cells. Thus, the gene can be mutated in mammalian cells and then transferred to bacteria for analysis of mutations. The advantage of this scheme is that in bacteria mutations can be rapidly and unambiguously scored. Furthermore, if lad is used as the mutational target, sophisticated bacterial molecular genetics can be applied to characterize the mutations (5, 6). In lad, an extensive deletion map allows precise localization of mutations by using genetic techniques. Moreover, accurate DNA sequence assignments can be made for nonsense mutations by using simple genetic techniques.The use of shuttle vectors for determination of mutagenic specificity has been limited by a high spontaneous mutation frequency associated with transfection (1-4) and by an inability of the vectors to replicate efficiently in human cells. Recently, both of these problems were solved by the discovery that the human cell line 293 (7) replicates simian virus 40 (SV40)-based shuttle vectors extremely well with a relatively low spontaneous mutation frequency (4). When 293 cells containing shuttle vectors are exposed to mutagens, an increase in mutation frequency above the spontaneous background is readily obtained. Therefore, induced mutations can be rapidly isolated and characterized.We demonstrate here the operation ofthe lacd shuttle using UV light as the mutagen. The DNA sequence changes for 53 UV light-induced point mutations, as well as for 32 point mutations from unmutagenized cells, are reported. The mutagenic specificity of UV light thereby obtained in human cells has close parallels with that obtained previously for UV light treatment of Escherichia coli. This result suggests that human and bacterial cells may react to UV light damage in similar ways. It also argues that the lacd shuttle system is indeed detecting true UV light-induced mutations. Thus, it appears that this experimental system can rapidly elucidate the outcome of a mutagen's interaction with DNA in human cells at the most fundamental genetic level, the DNA sequence change. MATERIALS AND METHODSVector DNA. The previously described (4) plasmid pJYMib was used as the shuttle vector. pJYMib contains all of SV40, pML (a pBR322 derivative), the entire lacI gene, and the amino-terminal portion of lacZ. Plasmid DNA was prepared by the alkaline lysis procedure and purified on cesium chloride gradients as described (8)....

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Section: Methodssupporting

confidence: 71%

The lacI shuttle: rapid analysis of the mutagenic specificity of ultraviolet light in human cells.

Lebkowski¹,

Clancy²,

Miller³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

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“…The presence of multiple mutations was also observed in the bacterial and phage systems in which mutants were characterized by DNA sequencing (8,11,15,36). As discussed above, multiple mutations would not be detected in the analysis of nonsense mutations, as in the lacI system.…”

Section: Resultsmentioning

confidence: 97%

“…filling would predict base changes at sites other than those of UV photoproducts. Thus, an analysis of the characteristics of UV-induced mutations should yield information on the mechanism of gap filling and mutation induction.The analysis of induced mutations in mammalian cells has been hampered by the lack of the elegant genetic systems that have been exploited for this purpose in bacterial cells and yeast (6,8,9,11,14,15,19,36). The use of shuttle vector plasmids that can replicate in both mammalian cells and bacterial cells should provide a solution to this problem.…”

mentioning

confidence: 99%

Sequence specificity of point mutations induced during passage of a UV-irradiated shuttle vector plasmid in monkey cells.

Hauser¹,

Seidman²,

Sidur³

et al. 1986

Mol. Cell. Biol.

170

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A simian virus 40-based shuttle vector was used to characterize UV-induced mutations generated in mammalian cells. The small size and placement of the mutagenesis marker (the supF suppressor tRNA gene from Escherichia cohl) within the vector substantially reduced the frequency of spontaneous mutations normally observed after transfection of mammalian cells with plasmid DNA; hence, UV-induced mutations were easily identified above the spontaneous background. UV-induced mutations characterized by DNA sequencing were found primarily to be base substitutions; about 56% of these were single-base changes, and 17% were tandem double-base changes. About 24% of the UV-induced mutants carried multiple mutations clustered within the 160-base-pair region sequenced. The majority (61%) of base changes were the G C-A -T transitions; the other transition (A * T-G C) and all four transversions occurred at about equal frequencies. Hot spots for UV mutagenesis did not correspond to hot spots for UV-induced photoproduct formation (determined by a DNA synthesis arrest assay); in particular, sites of TT dimers were underrepresented among the UV-induced mutations. These observations suggest to us that the DNA polymerase(s) responsible for mutation induction exhibits a localized loss of fidelity in DNA synthesis oi UV-damaged templates such that it synthesizes past UV photoproducts, preferentially inserting adenine, and sometimes misincorporates bases at undamaged sites nearby.Recent studies on viral and cellular oncogenes have provided strong evidence that mutations can play a fundamental role in cellular transformation and carcinogenesis (3). In addition, many heritable diseases and developmental anomalies have a mutational origin. Although numerous mutagenic agents are known to interact with DNA, the molecular mechanisms by which these agents may cause mutations in mammalian cells are not well understood. To approach this problem, we have used a UV-irradiated simian virus 40 (SV40)-based shuttle vector to analyze the sequence specificities of point mutations induced in mammalian cells by DNA damage. We reasoned that a detailed knowledge of the characteristics and spectrum of the mutations formed would provide information about the molecular mechanisms of mutation induction.In our previous work on the replication of UV-damaged SV40 DNA in mammalian cells (1,32,34), we demonstrated that replication of damaged templates appears to occur by a dimer-bypass mechanism in which replication forks proceed beyond UV-induced lesions, leaving gaps (single-strand discontinuities of 50 to 150 bases) in the daughter strands. Replication is completed at the normal terminus, and relaxed circular gapped molecules are produced. These gapped molecules are then slowly converted to SV40 form I DNA. We proposed that these single-strand gaps would be filled by DNA synthesis which might include insertion of mismatched bases at the sites of UV-induced lesions in the template strands, leading to the induction of point mutations at the damage sites. An alternat...

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“…Tandem double CC -* TT mutations have been thought to be a specific indicator of UV damage to DNA and have been detected after UV-irradiation in bacteria (22)(23)(24), yeast (25), and primate cells (26). This mutation has also been identified in the p53 gene of squamous cell skin carcinomas of human subjects (11), presumably as a result of UV exposure.…”

Section: Discussionmentioning

confidence: 99%

Tandem double CC-->TT mutations are produced by reactive oxygen species.

Reid

Loeb

1993

Proc. Natl. Acad. Sci. U.S.A.

134

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Oxidative damage to DNA is mutagenic and thus may play a role in carcinogenesis. Because of the large number of different DNA lesions formed by oxidative species, no genetic alteration so far identified is exclusively associated with oxygen damage. Tandem double CC --TT mutations are known to occur via UV damage to DNA and are thought to be a specific indicator of UV exposure. Using a sensitive reversion assay that can detect both single and double mutations within the same codon of the M13-encoded lacZa gene, we show that treatments that produce reactive oxygen species can also produce tandem double CC -k TT mutations. The frequency at which these mutations occur is less than that for single base mutations by a factor of approximately 30. The induction of these mutations is inhibited by treatment that scavenges hydroxyl radicals. This unique mutation provides a marker of oxygen free radical-induced mutagenesis in cells that are not exposed to UV-irradiation and an indicator for assessing the involvement of oxidative damage to DNA in aging and tumor progression.

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Changes in DNA base sequence induced by targeted mutagenesis of lambda phage by ultraviolet light

Cited by 138 publications

References 37 publications

The lacI shuttle: rapid analysis of the mutagenic specificity of ultraviolet light in human cells.

The lacI shuttle: rapid analysis of the mutagenic specificity of ultraviolet light in human cells.

Sequence specificity of point mutations induced during passage of a UV-irradiated shuttle vector plasmid in monkey cells.

Tandem double CC-->TT mutations are produced by reactive oxygen species.

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