Chemical synthesis and translesion replication of a cis-syn cyclobutane thymine-uracil dimer

Abstract

The cytosine base in DNA undergoes hydrolytic deamination at a considerable rate when UV radiation induces formation of a cyclobutane pyrimidine dimer (CPD) with an adjacent pyrimidine base. We have synthesized a phosphoramidite building block of a cis-syn cyclobutane thymine-uracil dimer (T[]U), which is the deaminated form of the CPD at a TC site, and incorporated it into oligodeoxyribonucleotides. The previously reported method for synthesis of the thymine dimer (T[]T) was applied, using partially protected thymidylyl-(3'-5')-2'-deoxyuridine as the starting material, and after triplet- sensitized irradiation, the configuration of the base moiety in the major product was determined by NMR spectroscopy. Presence of the cis-syn cyclobutane dimer in the obtained oligonucleotides was confirmed by UV photoreversal and reaction with T4 endonuclease V. Using a 30mer containing T[]U, translesion synthesis by human DNA polymerase eta was analyzed. There was no difference in the results between the templates containing T[]T and T[]U and pol eta bypassed both lesions with the same efficiency, incorporating two adenylates. This enzyme showed fidelity to base pair formation, but this replication causes a C-->T transition because the original sequence is TC.

Scheme 1. Synthesis of a phosphoramidite building block of the cis–syn cyclobutane thymine–uracil dimer. (i) 80% AcOH, room temperature, 2 h; (ii) hν, CH₃CN–H₂O–acetone (15/4/1 v/v/v), 4°C, 10 h; (iii) DMT-Cl, pyridine, room temperature, 3 h; (iv) NH₂NH₂·H₂O, pyridine–AcOH (4/1 v/v), room temperature, 5 min; (v) [(CH₃)₂CH]₂NP(Cl)OCH₂CH₂CN, [(CH₃)₂CH]₂NC₂H₅, THF, room temperature, 30 min.

Scheme 1. Synthesis of a phosphoramidite building block of the cis–syn cyclobutane thymine–uracil dimer. (i) 80% AcOH, room temperature, 2 h; (ii) hν, CH₃CN–H₂O–acetone (15/4/1 v/v/v), 4°C, 10 h; (iii) DMT-Cl, pyridine, room temperature, 3 h; (iv) NH₂NH₂·H₂O, pyridine–AcOH (4/1 v/v), room temperature, 5 min; (v) [(CH₃)₂CH]₂NP(Cl)OCH₂CH₂CN, [(CH₃)₂CH]₂NC₂H₅, THF, room temperature, 30 min.

Figure 1

Reversed phase HPLC analyses of the reaction mixture after the triplet-sensitized photoreaction for 10 h (A), the fractions collected after purification of compound 3 by silica gel chromatography (B) and the same sample as (B) after treatment with ammonia water at room temperature for 2 h (C). The acetonitrile gradient was 11–19% (A and B) or 0–10% (C) for 20 min.

Figure 2

(A) Reversed phase HPLC analysis of a crude sample of the T[]U-containing 30mer, using a gradient of 7–13% acetonitrile for 20 min. (B) Anion exchange HPLC analysis of the 30mer after purification by reversed phase HPLC, using a gradient of 0.4–1.2 M ammonium formate for 20 min.

Figure 3

Reversed phase HPLC analysis to confirm the presence of T[]U in the synthesized oligonucleotide. The purified 8mer, d(GTAT[]UATG) (A), a UV-irradiated sample of the T[]U 8mer (B) and the undamaged, uracil-containing 8mer, d(GTATUATG) (C) were analyzed using a 5–13% acetonitrile gradient for 20 min.

Figure 4

T4 endonuclease V reaction of a 30mer duplex containing T[]U. The 5′-³²P-labeled undamaged (lanes 1 and 2) and T[]T- (lanes 3–10) and T[]U-containing (lanes 11–18) 30mers were hybridized to a 3-fold excess of the complementary 30mer and incubated with T4 endonuclease V at 37°C. The products after the indicated times were subjected to PAGE under denaturing conditions.

Figure 5

Comparison of translesion synthesis past T[]T and T[]U. (A) The 5′-³²P-labeled 14mer was hybridized to the undamaged TT 30mer template (lanes 1–5) or the template containing T[]T (lanes 6–10) or T[]U (lanes 11–15) and incubated with increasing amounts of pol η or pol α in the presence of four dNTPs. The amounts of pol η were 0.56 (lanes 2, 7 and 12), 1.7 (lanes 3, 8 and 13) and 5.0 fmol (lanes 4, 9 and 14) and the amount of pol α was 5.0 fmol (lanes 5, 10 and 15). Lanes 1, 6 and 11 contained no enzyme. The products were subjected to PAGE under denaturing conditions. (B) The 5′-³²P labeled 16mer primer was hybridized to the undamaged 30mer template (lanes 1–6) or the template containing T[]T (lanes 7–12) or T[]U (lanes 13–18) and incubated with pol η (1.0 fmol), in the presence of four dNTPs (lanes 2, 8 and 14) or one of the indicated dNTPs (lanes 3–6, 9–12 and 15–18) or in the absence of dNTP (lanes 1, 7 and 13).