Detection of Transcription of Plasmid Sequences in Human Cells

To determine whether the sequence containing the 8-oxoG lesion is transcribed in each shuttle vector plasmid used, a standard protocol for reverse transcription-polymerase chain reaction (RT-PCR) is employed. Different monomodified plasmids (pSGO: C, pS AoriGO: C, pS AplateC: GO, and pS ApearlyGO: C) are trans-fected into normal, nontransformed human cell strain 198VI, derived from a skin biopsy of a normal child, in which there is no TAg sequence expressed. After

12 hr, total RNA is prepared by the guanidium thiocyanate method15 and treated

13 J. Wagner, H. Kamiya, and R. P. Fuchs, J. Mol. Biol. 265, 302 (1997).

14 F. Le Page, A. Klungland, D. E. Barnes, A. Sarasin, and S. Boiteux, Proc. Natl. Acad. Sci. U.S.A.

15 P. Chomczynski and N. Sacchi, Anal. Biochem. 162, 156 (1987).

with DNase I to eliminate any residual plasmid DNA. Aliquots of isolated mRNA are analyzed for their quality by agarose gel electrophoresis. Five micrograms of total RNA is reverse transcribed by incubation for 30 min at 37° in a final vo ume of 50 ¡i\ containing 1000 U of Moloney murine leukemia virus (Mo-MuLV) reverse transcriptase (GIBCO-BRL), 1 x RT buffer [50 mM Tris-HCl (pH 8.3), 75 mMKCl, 3 mMMgCl2], dNTPs (1 mMeach; (Pharmacia), 4 mMdithiothreitol (DTT; GIBCO-BRL), RNase inhibitor (1 U/ml; Boehringer), and 1 ßg of random hexamers (Pharmacia). The reaction is stopped by incubation for 5 min at 95°. PCR amplifications are performed with an automatic thermocycler (PerkinElmer, Courtaboeuf, France) in the presence of 2 U of Taq polymerase (AmpliTaq; PerkinElmer), 10 pmol of each sense and antisense primer of 25 bp, and PCR beads containing reaction buffer [10 mM Tris-HCl (pH 8.3), 5 mM KCl, 0.01% (w/v) gelatin], dNTPs (250 ßM each), and 1 mM MgCl2 (Pharmacia Biotech)]. The primers used, which amplify a 270-bp sequence, are complementary to the end of the coding sequence of the TAg gene and to the 3' end of the oligonucleotide carrying the lesion and are, respectively, 5'-AAAATGAATGCAATTGTTGT-3' and 5'-CTTGAGCGTCGATTTTTGTG-3'. After 1 cycle at 95° for 2 min, 30 cycles of denaturation, annealing, and extension are performed at 95° (1 min), 60° (1 min), and 72° (2 min), respectively. After 10 min at 12° to ensure that the final extension step is complete, aliquots of the products are analyzed by electrophoresis on a 1.5% (w/v) agarose gel and the DNA is extracted from the gel with a Nucleo-trap kit (Macherey Nagel, Düren, Germany). Sequencing of the amplified DNA fragments is carried out by the chain termination method, using a double-stranded DNA cycle sequencing system kit (GIBCO-BRL).

This approach has allowed us to verify that the absence of the SV40 early promoter prevents transcription of the oligonucleotide containing the GO lesion.

One possible explanation for the inability of cells that lack TCR to remove 8-oxoG in a transcribed sequence despite their capacity to repair 8-oxoG in a nontranscribed sequence is that access of repair enzymes to the lesion is prevented by a stalled RNA polymerase II (RNAPII). The classic model for TCR is that only lesions that block RNAP will be subject to preferential repair, and by extension that inability to perform TCR should result in an extended block to transcription. We therefore use RT-PCR to compare transcription 12 hr after transfection of the sequence containing the 8-oxoG in normal cells and TCR-defective cells, which do not remove the lesion. Transcription of the opposite strand does not interfere with the assay, because it occurs from the late promoter and hence later in time than transcription of TAg and thus of the lesion-containing sequence located in its 3' UTR.

To determine whether the lesion blocks transcription in cells defective in its repair, the RT-PCR analysis is performed on RNA extracted from diploid, nontransformed repair-defective cells isolated from a xeroderma pigmentosum group G patient also exhibiting Cockayne syndrome (XP-G/CS patient: XPCS1LV

Fig. 4. Blockage of transcription in vivo at a plasmid sequence containing the 8-oxoG lesion. RT-PCR analysis of samples collected 12 hr after transfection of the nonreplicative shuttle vector pSAoriGO: C into primary diploid cells isolated from a normal individual (198VI) or primary diploid cells isolated from a xeroderma pigmentosum group G patient also exhibiting Cockayne syndrome (XPCS1LV). Transcription of the TAg gene is indicated by the 300-bp product from primers located near the 5' end of the gene. Transcription of the sequence containing the 8-oxoG is revealed by the presence of a 270-bp product from primers spanning the site of the lesion. A 3-fold excess of product was loaded in the XP-G/CS cells to emphasize the absence of the 270-bp band. The left lane contains size markers. [Reproduced from F. Le Page, E. E. Kwoh, A. Avrutskaya, A. Gentil, S. A. Leadon, A. Sarasin, and P. K. Cooper, Cell 101, 159 (2000), with permission.]

Fig. 4. Blockage of transcription in vivo at a plasmid sequence containing the 8-oxoG lesion. RT-PCR analysis of samples collected 12 hr after transfection of the nonreplicative shuttle vector pSAoriGO: C into primary diploid cells isolated from a normal individual (198VI) or primary diploid cells isolated from a xeroderma pigmentosum group G patient also exhibiting Cockayne syndrome (XPCS1LV). Transcription of the TAg gene is indicated by the 300-bp product from primers located near the 5' end of the gene. Transcription of the sequence containing the 8-oxoG is revealed by the presence of a 270-bp product from primers spanning the site of the lesion. A 3-fold excess of product was loaded in the XP-G/CS cells to emphasize the absence of the 270-bp band. The left lane contains size markers. [Reproduced from F. Le Page, E. E. Kwoh, A. Avrutskaya, A. Gentil, S. A. Leadon, A. Sarasin, and P. K. Cooper, Cell 101, 159 (2000), with permission.]

cell strain) in comparison with normal 198VI cells 12 hr after transfection of pSAoriGO: C. The primers described above are used to amplify a 270-bp region containing the lesion. Transcription of sequences upstream of the lesion is also examined, as a positive control, by coamplification with another pair of primers (20 bp), 5'-GGAGGCTTCTGGGATGCAAC-3' and 5'-GAGCTTTAAATCTCTG TAGG-3', chosen to amplify a 300-bp coding region near the 5' end of the TAg gene. Transcription of the coding sequence of the TAg gene in the repair-deficient cells is compared with control. Whereas both cell strains transcribe TAg efficiently, only the normal cells transcribe the 8-oxoG-containing sequence (Fig. 4). No transcript that spans the lesion is detectable in repair-defective cells despite the occurrence of active transcription upstream of the lesion.

This technique allows us to correlate the state of transcription of the DNA sequences containing the lesion and the level of its repair on the transcribed strand. Our results strongly suggest that TCR of the oxidative lesion 8-oxoG is associated with in vivo blockage of RNA polymerase II at the lesion site.

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