Synthesis and mutagenesis of the butadiene-derived N3 2'-deoxyuridine adducts

Abstract

1,3-Butadiene is a known carcinogen and mutagen that acts through a variety of metabolic intermediates that react with DNA, forming stable and unstable lesions on dG, dA, dC, and dT. The N3 2'-deoxyuridine adducts are a highly stable, stereoisomeric mixture of adducts derived from the reaction of cytosine with the monoepoxide metabolite of butadiene, followed by spontaneous deamination. In this study, the phosphoramidites and subsequent oligodeoxynucleotides containing the N3 2'-deoxyuridine adducts have been constructed and characterized. Using a single-stranded shuttle vector DNA, the mutagenic potential of these adducts has been tested following replication in mammalian cells. Replication past the N3 2'-deoxyuridine adducts was found to be highly mutagenic with an overall mutation yield of approximately 97%. The major mutations that were observed were C to T transitions and C to A transversions. In vitro, these adducts posed a complete block to both the Klenow fragment of Escherichia coli polymerase I and polymerase epsilon, while these lesions significantly blocked polymerase delta. These data suggested a possible involvement of bypass polymerases in the in vivo replication of these lesions. Overall, these findings indicate that the N3 2'-deoxyuridine adducts are highly mutagenic lesions that may contribute to butadiene-mediated carcinogenesis.

Figure 1

Analysis of 12-mer oligodeoxynucleotide 5′-GCTAGCXAGTCC-3′. (A) Capillary gel electrophoresis profile. (B) HPLC profile of the enzymatic hydrolysate. The HPLC trace of an authentic sample of the adducted nucleoside is superimposed.

Figure 2

MALDI-TOF analysis of partial exonuclease digestion of 12-mer oligodeoxynucleotide 5′-GCTAGCXAGTCC-3′. (A) 3′→ 5′ sequencing using phosphodiesterase I. (B) 5′→ 3′ sequencing using phosphodiesterase II.

Figure 3

Autoradiographs displaying the result of mutagenic replication past the BD N3-dU adducts in COS-7 mammalian cells. Single-stranded pMS2 vector containing the BD N3-dU adducts or control dC was transfected into COS-7 cells and replicated for 48 h. The progeny plasmids were then recovered from the COS-7 cells and used to transform DH5α E. coli cells. Individual transformants were picked and grown in 96-well plates containing LB with ampicillin. The resultant colonies were transferred in four replicates onto Whatman 541 filter papers, one for each of the four probes G, C, A, and T, and were differentially hybridized.

Figure 4

Primer extension reactions catalyzed by Kf on the BD N3-dU adducted template. Control dC or BD N3-dU adducted 32-mer DNA templates were annealed to a -2 primer. The DNA substrates (5 nM) were incubated in the presence of all four dNTPs (100 μM) without or with increasing concentrations (0.0005, 0.005, 0.05 and 0.5 units) of Kf (lanes 2-5 and 7-10) for 15 min at room temperature. The position of the 14-nucleotide primer is indicated. U* indicates the position of the modified uracil on the template.

Figure 5

Primer extension reactions catalyzed by calf thymus pol δ and human pol ε on the BD N3-dU adducted template. Control dC or BD N3-dU adducted 32-mer DNA templates were annealed to a -2 primer. (A) The DNA substrates (5 nM) were incubated in the presence of all four dNTPs (100 μM) without or with increasing concentrations (0.008, 0.08 and 0.8 units) of pol δ, in the presence of 70 ng of calf thymus PCNA for 30 min at room temperature. (B) Time course experiments (at time intervals of 0, 1, 5,10, 15, 20 and 30 min) were carried out with the DNA substrates (5 nM) at room temperature in the presence of all four dNTPs (100 μM) with 0.1 units of pol ε. The positions of the 14-nucleotide primers and the 32-nucleotide full-length products are indicated. U* indicates the position of the modified uracil on the template.

Scheme 1

Synthesis of 5′-DMT-3′ p-Tol-2′deoxyuridine

Scheme 2

Synthesis of 2-O-t-butyldimethylsilyl-3-butene-1-(p-toluenesulfonate)

Scheme 3

Synthesis of 5′-DMT-N3-(3-buten-2-O-t-butyldimethylsilyl-1yl)-2′ deoxyuridine-3′-O-(N,N,-diisopropyl-O-cyanoethyl)-phosphoramidite