Abnormal ultraviolet mutagenic spectrum in plasmid DNA replicated in cultured fibroblasts from a patient with the skin cancer-prone disease, xeroderma pigmentosum.

Seetharam, Saraswathy; Protić-Sabljić, M; Seidman, Michael M.; Kraemer, Kenneth H.

doi:10.1172/jci113248

Cited by 48 publications

(9 citation statements)

References 13 publications

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“…Site 136, which is located in the middle of a run of three ART base pairs, was the strongest hot spot, with 11% of the total base substitutions found there. This hot spot is unique to plasmids replicated in XP-V cells; i.e., to our knowledge, it has not previously been found with UVirradiated supF genes that have been replicated in mammalian cells (17,24,25). All the mutations at this site were transversions, whereas those at the other four hot spots were mainly transitions.…”

Section: Resultsmentioning

confidence: 61%

Xeroderma pigmentosum variant cells are less likely than normal cells to incorporate dAMP opposite photoproducts during replication of UV-irradiated plasmids.

Wang

Maher

McCormick³

1991

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

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Xeroderma pigmentosum (XP) variant patients show the clinical characteristics of the disease, with increased frequencies of skin cancer, but their cells have a normal, or nearly normal, rate of nucleotide excision repair of UV-induced DNA damage and are only slightly more sensitive than normal cells to the cytotoxic effect of UV radiation. However, they are significantly more sensitive to its mutagenic effect. To examine the mechanisms responsible for this hypermutability, we transfected an XP variant cell line with a UV-irradiated (at 254 nm) shuttle vector carrying the supF gene as a target for mutations, allowed replication of the plasmid, determined the frequency and spectrum of mutations induced, and compared the results with those obtained previously when irradiated plasmids carrying the same target gene It is now widely recognized that the transformation of normal cells into tumorigenic cells is a multistep process, and substantial evidence indicates that mutations play a fundamental role in cellular transformation and carcinogenesis, as well as in many inheritable diseases and developmental anomalies (1, 2). However, our understanding of the factors and influences governing the formation of these changes in gene structure is considerably less advanced. Cells isolated from patients with the rare autosomal recessive disorder xeroderma pigmentosum (XP) present a unique model system for investigating DNA repair and mutagenesis in human cells. In the present study, we made use ofa shuttle vector assay to investigate the kinds of mutations induced when a UV-irradiated plasmid replicated in cells derived from the class of XP patients called XP variants in order to provide clues to the mechanisms responsible for the hypermutagenic effect of UV radiation on these cells. XP variants inherit the characteristic predisposition to sunlight-induced skin cancer, but unlike the majority of XP patients, their cells do not exhibit a significant deficiency in the rate of nucleotide excision repair of endogenous UVinduced DNA damage, including both cyclobutane pyrimidine dimers (3-8) and pyrimidine-pyrimidone(6-4) photoproducts (7, 8). Cells from XP variant patients have an abnormality in the manner in which DNA replicates on a template containing UV lesions (9-11) and an inability to convert a very minor UV photoproduct to an excisable lesion (12). They are only slightly more sensitive than cells from normal donors to the cytotoxic effect of UV but are significantly more sensitive to its mutagenic action (13-15). However, the molecular mechanism(s) responsible for the abnormal sensitivity of XP variant cells to UV-induced mutations has not been explained.To examine this question, we UV-irradiated a shuttle vector, pS189 (16), carrying the supF gene as the target for mutations and transfected the plasmids into a simian virus 40-transformed XP variant cell line (XP-V) where they could be replicated by the human cell polymerase(s). The progeny plasmids were analyzed for the frequency of supF mutants and the kinds of mu...

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Section: Resultsmentioning

confidence: 61%

Xeroderma pigmentosum variant cells are less likely than normal cells to incorporate dAMP opposite photoproducts during replication of UV-irradiated plasmids.

Wang

Maher

McCormick³

1991

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

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“…exonucleases) or the activity of a defective DNA ligase in BS cells introduced more mutations at the joining sites than in normal cells. Point mutations may reflect defective functioning of the excision repair system which contains a ligase step, or action of an error-prone polymerase entering at a DNA strand break, as proposed previously for pZ 189 in xeroderma pigmentosum group A cells (Seidman et al, 1987).…”

Section: Discussionmentioning

confidence: 80%

“…In order to study DNA ligase activity in vivo, we developed a host cell ligation (end-joining) assay analogous to plasmid DNA repair host cell reactivation assays (Protic-Sabljic et al, 1985;Bredberg et al, 1986;Seidman et al, 1987), using the replicating shuttle vector plasmid pZ 189 (Seidman et al, 1985). Linear plasmid DNA was produced by digestion with restriction endonucleases and then transfected into fibroblast or lymphoblast host cells.…”

Section: Introductionmentioning

confidence: 99%

Joining of linear plasmid DNA is reduced and error-prone in Bloom's syndrome cells.

Rünger

Kraemer

1989

The EMBO Journal

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A linearized, replicating, shuttle vector plasmid, pZ189, was used to measure in vivo DNA joining ability of cells from patients with the cancer‐prone, immunodeficient, chromosome breakage disorder, Bloom's syndrome (BS). The BS cell lines we studied were reported to contain reduced in vitro activity of DNA ligase I. We assessed in vivo joining ability by transfecting linear plasmids with overlapping or blunt ends (produced by EcoRI or StuI) into BS and normal fibroblast or lymphoblast host cells and measuring the amount of re‐joined, replicated plasmids by their ability to transform bacteria. With plasmids having either overlapping or blunt ends we found a 1.3‐ to 3‐fold lower (P less than 0.05) joining efficiency in BS cells than in the normal cells. The mutation frequency of the recovered plasmids was measured by screening for function of the suppressor tRNA contained in pZ189, for plasmid size, for presence of restriction sites, or by DNA sequencing. The spontaneous mutation frequency with the circular plasmid was 0.05‐0.08% with both BS cell lines, values 2‐ to 21‐fold higher (P less than 0.03) than with the normal cell lines. The mutation frequency with the linear plasmid passaged through both BS cell lines was 21‐52%, values 1.4‐ to 5.4‐fold higher (P less than 0.001) than with the normal lines. Detailed analysis of 210 recovered plasmids revealed an increase (P less than or equal to 0.001) in deletions, insertions or complex mutations at the joining sites, and in point mutations with the EcoRI cut plasmid with the BS cells in comparison to the normal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…As soon as the plasmid pZ189 became available (5), a series of studies to elucidate the mutation spectrum of ultraviolet light (UV) in human cells were carried out by Kraemer and colleagues (22)(23)(24)(25). UV was theˆrst subject of the studies because UV-induced DNA damage, cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4 PPs), had been well characterized and the DNA repair-deˆcient cells derived from xeroderma pigmentosum (XP) patients with 7 genetic complementation groups were by then available.…”

Section: Application Of the Shuttle Vector Plasmids To Uv Mutagenesismentioning

confidence: 99%

“…As a result of various studies on UV-induced mutations in the shuttle vector plasmids introduced into normal human and XP cells (22)(23)(24)(25)(26)(27), some keyˆndings that are now common knowledge were obtained. The G:C to A:T transition is the major type of UV-induced mutation, and its frequency is higher in DNA repair-deˆcient XP cells than in normal human cells.…”

Section: Application Of the Shuttle Vector Plasmids To Uv Mutagenesismentioning

confidence: 99%

The Achievement of Shuttle Vector Techniques in Mammalian Cell Mutation Research

Yagi¹

2013

Genes and Environment

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Shuttle vector plasmids have made major contributions to the advancement of mutation research for the last two decades. I have been involved in the development of shuttle vector plasmids and their experimental protocols, and herein provide a chronicle of shuttle vector studies. In the late 1960s, in vitro mammalian cell culture became a common technique in cell biology. Geneticists established a method to detect gene mutation as 8-azaguanine or 6-thioguanine resistance after cells had been exposed to radiation or chemicals. The resistance of the cells was found to be due to a mutation of the hypoxanthine-guanine phosphoribosyl transferase gene; however, which base was changed in the gene remained di‹cult to determine until DNA sequencing techniques were developed. After the Sanger method of DNA sequencing became available to geneticists, the next issue was how to identify numerous base changes in mutants easily and rapidly. A breakthrough on this issue was the use of the shuttle vector plasmid pZ189, which was developed for practical applications by Seidman and colleagues. Along with the development of new molecular biology techniques such as polymerase chain reaction and automatic DNA sequencing, pZ189 has been modiˆed to several forms as adaptations to the new techniques. These shuttle vector plasmids remain useful tools to reveal what types of mutations are induced by newly identiˆed mutagens at the DNA sequence level. This article describes some of the contents of my JEMS award lecture in 2011.

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Abnormal ultraviolet mutagenic spectrum in plasmid DNA replicated in cultured fibroblasts from a patient with the skin cancer-prone disease, xeroderma pigmentosum.

Cited by 48 publications

References 13 publications

Xeroderma pigmentosum variant cells are less likely than normal cells to incorporate dAMP opposite photoproducts during replication of UV-irradiated plasmids.

Xeroderma pigmentosum variant cells are less likely than normal cells to incorporate dAMP opposite photoproducts during replication of UV-irradiated plasmids.

Joining of linear plasmid DNA is reduced and error-prone in Bloom's syndrome cells.

The Achievement of Shuttle Vector Techniques in Mammalian Cell Mutation Research

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