In vitro ligation of oligodeoxynucleotides containing C8-oxidized purine lesions using bacteriophage T4 DNA ligase

Abstract

Ligases conduct the final stage of repair of DNA damage by sealing a single-stranded nick after excision of damaged nucleotides and reinsertion of correct nucleotides. Depending upon the circumstances and the success of the repair process, lesions may remain at the ligation site, either in the template or at the oligomer termini to be joined. Ligation experiments using bacteriophage T4 DNA ligase were carried out with purine lesions in four positions surrounding the nick site in a total of 96 different duplexes. The oxidized lesion 8-oxo-7,8-dihydroguanosine (OG) showed, as expected, that the enzyme is most sensitive to lesions on the 3' end of the nick compared to the 5' end and to lesions located in the intact template strand. In general, substrates containing the OG.A mismatch were more readily ligated than those with the OG.C mismatch. Ligations of duplexes containing the OA.T base pair (OA = 8-oxo-7,8-dihydroadenosine) that could adopt an anti-anti conformation proceeded with high efficiencies. An OI.A mismatch-containing duplex (OI = 8-oxo-7,8-dihydroinosine) behaved like OG.A. Due to its low reduction potential, OG is readily oxidized to secondary oxidation products, such as the guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) nucleosides; these lesions also contain an oxo group at the original C8 position of the purine. Ligation of oligomers containing Gh and Sp occurred when opposite A and G, although the overall ligation efficiencies were much lower than those of most OG base pairs. Steady-state kinetic studies were carried out for representative examples of lesions in the template. Km increased by 90-100-fold for OG.C-, OI.C-, OI.A-, and OA.T-containing duplexes compared to that of a G.C-containing duplex. Substrates containing Gh.A, Gh.G, Sp.A, and Sp.G base pairs exhibited Km values 20-70-fold higher than that of the substrate containing a G.C base pair, while the Km value for OG.A was 5 times lower than that for G.C.

Figure 1

Structures of 8-oxo-7,8-dihydro-2′-deoxyguanosine (OG), 8-oxo-7,8-dihydro-2′-deoxyinosine (OI) and 8-oxo-7,8-dihydro-2′ deoxyadenosine (OA).

Figure 2

A: Oxidation from G to Gh and Sp can occur sequentially via OG or in one step using a 4-eoxidant such as singlet oxygen. B: Oxidation of OG with one-electron oxidants leads to Gh and Sp.

Figure 3

Sequences used for Template/Primer1/Primer2 duplexes.

Figure 4

Ligation with lesions in the template opposite the 3′ end of the nick. G, OG, Gh and Spcontaining oligomers were each base paired with C, A, T, and G in the opposite strand. Lanes without ligation products were control studies with no enzyme added. Sequences used are indicated in Figure 3a.

Figure 5

Ligation efficiencies of oligodeoxynucleotides containing lesions X=OG, OI, OA, Gh or Sp around the nick; X = lesion; N = C, A, T, G. A: lesions in the template opposite the 3′ end of the nick; B: lesions on the 3′ end of the nick; C: lesions in the template opposite the 5′ end of the nick; D: lesions on the 5′ end of the nick. DNA sequences used in Figure 5A, 5B, 5C and 5D are indicated in Figure 3a, 3b, 3c and 3d respectively. Primer1 shown in red was 5′-end labeled for PAGE analysis. Asterisks (panels C and D) indicate a comparison of 5′-T:G and 5′-Gh:G, both of which are ligated with high efficiency.

Figure 6

Base pairing of OG, OI, OA, Gh and Sp. OG can base pair with C at an anti-anti conformation and with A at a syn-anti conformation. OI can form a similar base pair with the same conformation as OG. OA can only form an anti-anti base pair with T. Gh is proposed to base pair with A and G in a syn-anti conformation. Sp is proposed to base pair with A or G in a syn-anti conformation or potentially with G in an anti-anti conformation (shown).