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Sequence-specific p53 gene damage by chloroacetaldehyde and its repair kinetics in Escherichia coli

100%

Kowalczyk P., Cieśla J. M., Saparbaev M., Laval J., Tudek B.

2006

vol. 53

issue 2

337-347

Oxidative stress and certain environmental carcinogens, e.g. vinyl chloride and its metabolite chloroacetaldehyde (CAA), introduce promutagenic exocyclic adducts into DNA, among them 1,N6-ethenoadenine (εA), 3,N4-ethenocytosine (εC) and N2,3-ethenoguanine (εG). We studied sequence-specific interaction of the vinyl-chloride metabolite CAA with human p53 gene exons 5-8, using DNA Polymerase Fingerprint Analysis (DPFA), and identified sites of the highest sensitivity. CAA-induced DNA damage was more extensive in p53 regions which revealed secondary structure perturbations, and were localized in regions of mutation hot-spots. These perturbations inhibited DNA synthesis on undamaged template. We also studied the repair kinetics of CAA-induced DNA lesions in E. coli at nucleotide resolution level. A plasmid bearing full length cDNA of human p53 gene was modified in vitro with 360 mM CAA and transformed into E. coli DH5α strain, in which the adaptive response system had been induced by MMS treatment before the cells were made competent. Following transformation, plasmids were re-isolated from transformed cultures 35, 40, 50 min and 1-24 h after transformation, and further subjected to LM-PCR, using ANPG, MUG and Fpg glycosylases to identify the sites of DNA damage. In adaptive response-induced E. coli cells the majority of DNA lesions recognized by ANPG glycosylase were removed from plasmid DNA within 35 min, while MUG glycosylase excised base modifications only within 50 min, both in a sequence-dependent manner. In non-adapted cells resolution of plasmid topological forms was perturbed, suggesting inhibition of one or more bacterial topoisomerases by unrepaired ε-adducts. We also observed delayed consequences of DNA modification with CAA, manifesting as secondary DNA breaks, which appeared 3 h after transformation of damaged DNA into E. coli, and were repaired after 24 h.

Inhibition of DNA repair glycosylases by base analogs and tryptophan pyrolysate, Trp-P-1.

86%

Speina E., Cieśla J. M., Grąziewicz M.-A., Laval J., Kazimierczuk Z., Tudek B.

2005

vol. 52

issue 1

167-178

DNA base analogs, 2,4,5,6-substituted pyrimidines and 2,6-substituted purines were tested as potential inhibitors of E. coli Fpg protein (formamidopyrimidine -DNA glycosylase). Three of the seventeen compounds tested revealed inhibitory properties. 2-Thioxanthine was the most efficient, inhibiting 50% of 2,6-diamino-4-hydroxy-5N-methyl-formamidopyrimidine (Fapy-7MeG) excision activity at 17.1 μM concentration. The measured Kgi was 4.44 ± 0.15 μM. Inhibition was observed only when the Fpg protein was first challenged to its substrate followed by the addition of the base analog, suggesting uncompetitive (catalytic) inhibition. For two other compounds, 2-thio- or 2-oxo-4,5,6-substituted pyrimidines, IC50 was only 343.3 ± 58.6 and 350 ± 24.4 μM, respectively. No change of the Fpg glycosylase activity was detected in the presence of Fapy-7MeG, up to 5 μM. We also investigated the effect of DNA structure modified by tryptophan pyrolysate (Trp-P-1) on the activity of base excision repair enzymes: Escherichia coli and human DNA glycosylases of oxidized (Fpg, Nth) and alkylated bases (TagA, AlkA, and ANPG), and for bacterial AP endonuclease (Xth protein). Trp-P-1, which changes the secondary DNA structure into non-B, non-Z most efficiently inhibited excision of alkylated bases by the AlkA glycosylase (IC50 = 1 μM). The ANPG, TagA, and Fpg proteins were also inhibited although to a lesser extent (IC50 = 76.5 μM, 96 μM, and 187.5 μM, respectively). Trp-P-1 also inhibited incision of DNA at abasic sites by the β-lyase activity of the Fpg and Nth proteins, and to a lesser extent by the Xth AP endonuclease. Thus, DNA conformation is critical for excision of damaged bases and incision of abasic sites by DNA repair enzymes.

Mutagenic specificity of imidazole ring-opened 7-methylpurines in m13mp18 phage DNA

86%

Tudek B., Grąziewicz M., Kazanova O., Zastawny T. H., Obtułowicz T., Laval J.

Acta Biochimica Polonica

1999

vol. 46

issue 3

785-799

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3 Acta Biochimica Polonica

1 2006

1 2005

1 1999

3 Laval J.

3 Tudek B.

2 Cieśla J. M.

1 Grąziewicz M.

1 Grąziewicz M.-A.

1 Kazanova O.

1 Kazimierczuk Z.

1 Kowalczyk P.

1 Obtułowicz T.

1 Saparbaev M.

1 Speina E.

1 Zastawny T. H.

Search results

Sequence-specific p53 gene damage by chloroacetaldehyde and its repair kinetics in Escherichia coli

Inhibition of DNA repair glycosylases by base analogs and tryptophan pyrolysate, Trp-P-1.

Mutagenic specificity of imidazole ring-opened 7-methylpurines in m13mp18 phage DNA