New synthetic substrates of mammalian nucleotide excision repair system

Abstract

DNA probes for the studies of damaged strand excision during the nucleotide excision repair (NER) have been designed using the novel non-nucleosidic phosphoramidite reagents that contain N-[6-(9-antracenylcarbamoyl)hexanoyl]-3-amino-1,2-propandiol (nAnt) and N-[6-(5(6)-fluoresceinylcarbamoyl)hexanoyl]-3-amino-1,2-propandiol (nFlu) moieties. New lesion-imitating adducts being inserted into DNA show good substrate properties in NER process. Modified extended linear nFlu- and nAntr-DNA are suitable for estimation of specific excision activity catalysed with mammalian whole-cell extracts. The following substrate activity range was revealed for the model 137-bp linear double-stranded DNA: nAnt-DNA ≈ nFlu-DNA > Chol-DNA (Chol-DNA--legitimate NER substrate that contains non-nucleoside fragment bearing cholesterol residue). In vitro assay shows that modified DNA can be a useful tool to study NER activity in whole-cell extracts. The developed approach should be of general use for the incorporation of NER-sensitive distortions into model DNAs. The new synthetic extended linear DNA containing bulky non-nucleoside modifications will be useful for NER mechanism study and for applications.

Figure 1.

The general scheme of the organic synthesis procedure. Reagents and conditions: (i) N-hydroxysuccinimide ester of TFA-NH-(CH₂)₅-COOH, Et₃N, DMF; (ii) DMTCl, pyridine; (iii) aqueous NH₃ (25%), pyridine; (iv) (a) 9-anthracenecarboxylic acid, N-hydroxybenzotriazole, DCC, CH₂Cl₂, Et₃N, (b) 5(6)-carboxyfluorescein-NHS ester, Et₃N, CH₂Cl₂; and (v) 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphodiamidite, diisopropylammonium tetrazolide, CH₂Cl₂.

Figure 2.

The non-nucleoside fragments of the modified DNA strand, containing N-[6-(9-antracenylcarbomoyl)hexanoyl]-3-amino-1,2-propandiol [nAnt, (A)], N-[6-(5(6)-fluoresceinylcarbomoyl)hexanoyl]-3-amino-1,2-propandiol [nFlu, (B)] or cholesterol residue [Chol, (C)].

Figure 3.

The scheme of NER in vitro assay. Green hexagon represents a bulky modification; red asterisk, radioactive label; dark blue and grey ovals, the sequence of specific and non-specific templates, respectively.

Figure 4.

The NER dual incision activity of CHO cells extract. The excision products were detected by annealing to the template followed by end-labelling using α-[³²P]-dCTP and Taq DNA polymerase. The reaction products were resolved on a 10% denaturing polyacrylamide gel. (A) The comparison between original (28) (lanes 1–3) and modified (lanes 4–6) in vitro NER assays. In contrast to the original method the presence of template 1 in reaction mixture during the incubation of the model DNA substrate with cell extract increases the observed signal intensity. Model DNAs were incubated for 30 min at 30°C with cell extracts prepared from CHO cells (20 nМ DNA, 1.6 mg/ml of extract proteins with or without 500 nM template 1). (B) Specific product accumulation. The 20 nМ model DNAs was incubated for 15–60 min at 30°C with 1.6 mg/ml of extract proteins and 500 nM template 1. (C) Time dependence of the specific excision products accumulation. Grey squares correspond to nFlu–DNA; black circles, nAnt–DNA. The maximal products accumulation was taken as 100% and was detected for samples incubated for 30 min. The subsequent incubation leads to non-specific products degradation. (D) Comparison of the results obtained with template 1 and template 2.

Figure 5.

(A) The NER dual incision activity of CHO cells extract. Model DNAs were incubated for 30 min at 30°C with cell extracts prepared from CHO cells (20 nM model DNA, 0.3 or 1.6 mg/ml of extract proteins and 500 nM template 1).The excision products, containing nFlu, nAnt or Chol, were detected by annealing to a template followed by end-labelling using α-[³²P]-dCTP and Taq DNA polymerase. The reaction products were resolved on a 10% denaturing polyacrylamide gel. Non-modified 137-bp DNA was used as a negative control; ³²P-labelled oligonucleotides were used as size marker (lane M). (B) Relative signal of the target products for non-modified, nFlu– and nAnt–DNA after incubation with different concentrations of the extract proteins.

Figure 6.

(A) The NER dual incision activity of cancer cells extracts. Model DNAs were incubated for 30 min at 30°C with cell extracts prepared from HeLa, C33A or SiHa cells (20 nМ DNA, 1.6 mg/ml of extract proteins and 500 nM template 1). The excision products were detected by annealing to template followed by end-labelling using α-[³²P]-dCTP and Taq DNA polymerase. The reaction products were resolved on a 10% denaturing polyacrylamide gel. Non-modified 137-bp DNA was used as a negative control; ³²P-labelled oligonucleotides were used as size marker. (B) Densitometric analysis of the lanes 5 (HeLa, left), 8 (C33A, middle) and 11 (SiHa, right).