DNA base sequence changes and sequence specificity of bromodeoxyuridine-induced mutations in mammalian cells.

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 coli) 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 on UV-damaged templates such that it synthesizes past UV photoproducts, preferentially inserting adenine, and sometimes misincorporates bases at undamaged sites nearby.

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Thymidine-induced mutations in mammalian cells: sequence specificity and implications for mutagenesis in vivo.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.89.7.2829 ◽

1992 ◽

Vol 89 (7) ◽

pp. 2829-2833 ◽

Cited By ~ 9

Author(s):

M. T. Kresnak ◽

R. L. Davidson

Keyword(s):

Mammalian Cells ◽

Sequence Specificity ◽

Induced Mutations

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

Molecular and Cellular Biology ◽

10.1128/mcb.6.1.277 ◽

1986 ◽

Vol 6 (1) ◽

pp. 277-285 ◽

Cited By ~ 112

Author(s):

J Hauser ◽

M M Seidman ◽

K Sidur ◽

K Dixon

Keyword(s):

Dna Synthesis ◽

Hot Spots ◽

Mammalian Cells ◽

Shuttle Vector ◽

Simian Virus 40 ◽

Simian Virus ◽

Mutation Induction ◽

Sequence Specificity ◽

Induced Mutations ◽

Double Base

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 coli) 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 on UV-damaged templates such that it synthesizes past UV photoproducts, preferentially inserting adenine, and sometimes misincorporates bases at undamaged sites nearby.

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