Relationship between DNA methylation and mutational patterns induced by a sequence selective minor groove methylating agent

Jack D. Kelly, Alberto Inga, Fa Xian Chen, Prasad Dande, Dharini Shah, Paola Monti, Anna Aprile, Philip A. Burns, Gina Scott, Angelo Abbondandolo, Barry Gold, Gilberto Fronza

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

Me-lex, a methyl sulfonate ester appended to a neutral N- methylpyrrolecarboxamide-based dipeptide, was synthesized to preferentially generate N3-methyladenine (3-MeA) adducts which are expected to be cytotoxic rather than mutagenic DNA lesions. In the present study, the sequence specificity for DNA alkylation by Me-lex was determined in the p53 cDNA through the conversion of the adducted sites into single strand breaks and sequencing gel analysis. In order to establish the mutagenic and lethal properties of Me-lex lesions, a yeast expression vector harboring the human wild-type p53 cDNA was treated in vitro with Me-lex, and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. The results showed that: 1) more than 99% of the lesions induced by Me-lex are 3-MeA; 2) the co-addition of distamycin quantitatively inhibited methylation at all minor groove sites; 3) Me-lex selectively methylated A's that are in, or immediately adjacent to, the lex equilibrium binding sites; 4) all but 6 of the 33 independent mutations were base pair substitutions, the majority of which (17/33; 52%) were AT-targeted; 5) AT → TA transversions were the predominant mutations observed (13/33; 39%); 6) 13 out of 33 (39%) independent mutations involved a single lex-binding site encompassing positions A600-602 and 9 occurred at position 602 which is a real Me-lex mutation hotspot (n = 9, p <10-6, Poisson's normal distribution). A hypothetical model for the interpretation of mutational events at this site is proposed. The present work is the first report on mutational properties of Me-lex. Our results suggest that 3-MeA is not only a cytotoxic but also a premutagenic lesion which exerts this unexpected property in a strict sequence-dependent manner.

Original languageEnglish
Pages (from-to)18327-18334
Number of pages8
JournalJournal of Biological Chemistry
Volume274
Issue number26
DOIs
Publication statusPublished - Jun 25 1999

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DNA Methylation
Mutation
Yeast
Complementary DNA
Yeasts
Binding Sites
Poisson Distribution
Methylation
methyl lexitropsin
Dipeptides
Normal Distribution
DNA
Alkylation
Normal distribution
Base Pairing
Esters
Substitution reactions
Genes
Gels

ASJC Scopus subject areas

  • Biochemistry

Cite this

Relationship between DNA methylation and mutational patterns induced by a sequence selective minor groove methylating agent. / Kelly, Jack D.; Inga, Alberto; Chen, Fa Xian; Dande, Prasad; Shah, Dharini; Monti, Paola; Aprile, Anna; Burns, Philip A.; Scott, Gina; Abbondandolo, Angelo; Gold, Barry; Fronza, Gilberto.

In: Journal of Biological Chemistry, Vol. 274, No. 26, 25.06.1999, p. 18327-18334.

Research output: Contribution to journalArticle

Kelly, JD, Inga, A, Chen, FX, Dande, P, Shah, D, Monti, P, Aprile, A, Burns, PA, Scott, G, Abbondandolo, A, Gold, B & Fronza, G 1999, 'Relationship between DNA methylation and mutational patterns induced by a sequence selective minor groove methylating agent', Journal of Biological Chemistry, vol. 274, no. 26, pp. 18327-18334. https://doi.org/10.1074/jbc.274.26.18327
Kelly, Jack D. ; Inga, Alberto ; Chen, Fa Xian ; Dande, Prasad ; Shah, Dharini ; Monti, Paola ; Aprile, Anna ; Burns, Philip A. ; Scott, Gina ; Abbondandolo, Angelo ; Gold, Barry ; Fronza, Gilberto. / Relationship between DNA methylation and mutational patterns induced by a sequence selective minor groove methylating agent. In: Journal of Biological Chemistry. 1999 ; Vol. 274, No. 26. pp. 18327-18334.
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abstract = "Me-lex, a methyl sulfonate ester appended to a neutral N- methylpyrrolecarboxamide-based dipeptide, was synthesized to preferentially generate N3-methyladenine (3-MeA) adducts which are expected to be cytotoxic rather than mutagenic DNA lesions. In the present study, the sequence specificity for DNA alkylation by Me-lex was determined in the p53 cDNA through the conversion of the adducted sites into single strand breaks and sequencing gel analysis. In order to establish the mutagenic and lethal properties of Me-lex lesions, a yeast expression vector harboring the human wild-type p53 cDNA was treated in vitro with Me-lex, and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. The results showed that: 1) more than 99{\%} of the lesions induced by Me-lex are 3-MeA; 2) the co-addition of distamycin quantitatively inhibited methylation at all minor groove sites; 3) Me-lex selectively methylated A's that are in, or immediately adjacent to, the lex equilibrium binding sites; 4) all but 6 of the 33 independent mutations were base pair substitutions, the majority of which (17/33; 52{\%}) were AT-targeted; 5) AT → TA transversions were the predominant mutations observed (13/33; 39{\%}); 6) 13 out of 33 (39{\%}) independent mutations involved a single lex-binding site encompassing positions A600-602 and 9 occurred at position 602 which is a real Me-lex mutation hotspot (n = 9, p <10-6, Poisson's normal distribution). A hypothetical model for the interpretation of mutational events at this site is proposed. The present work is the first report on mutational properties of Me-lex. Our results suggest that 3-MeA is not only a cytotoxic but also a premutagenic lesion which exerts this unexpected property in a strict sequence-dependent manner.",
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AU - Inga, Alberto

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AU - Dande, Prasad

AU - Shah, Dharini

AU - Monti, Paola

AU - Aprile, Anna

AU - Burns, Philip A.

AU - Scott, Gina

AU - Abbondandolo, Angelo

AU - Gold, Barry

AU - Fronza, Gilberto

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