RNA polymerase II bypass of oxidative DNA damage is regulated by transcription elongation factors

Abstract

Oxidative lesions represent the most abundant DNA lesions within the cell. In the present study, we investigated the impact of the oxidative lesions 8-oxoguanine, thymine glycol and 5-hydroxyuracil on RNA polymerase II (RNA pol II) transcription using a well-defined in vitro transcription system. We found that in a purified, reconstituted transcription system, these lesions block elongation by RNA pol II to different extents, depending on the type of lesion. Suggesting the presence of a bypass activity, the block to elongation is alleviated when transcription is carried out in HeLa cell nuclear extracts. By purifying this activity, we discovered that TFIIF could promote elongation through a thymine glycol lesion. The elongation factors Elongin and CSB, but not TFIIS, can also stimulate bypass of thymine glycol lesions, whereas Elongin, CSB and TFIIS can all enhance bypass of an 8-oxoguanine lesion. By increasing the efficiency with which RNA pol II reads through oxidative lesions, elongation factors can contribute to transcriptional mutagenesis, an activity that could have implications for the generation or progression of human diseases.

Figure 1

Bypass of oxidative lesions. (A) The transcription template contains a single oxoG-, Tg- or OHdU-lesion located 488/489 bp downstream of the AdMLP promoter start site. The oxidative lesion abolishes the ApaLI restriction site. (B) In vitro transcription of oxidative lesion containing template on the transcribed strand using either RTS (lanes 1–5), or HeLa NE (lanes 6–10) or on the non-transcribed strand (NTS) using RTS (lanes 11–13). Quantitative data (mean±s.d.) are derived from at least three independent experiments. Arrest (489 nt) and bypass (525 nt) transcripts are indicated. (C) Glycosylase assays (as described by Shimizu et al (2003) (lanes 1–3) and transcription assays (lanes 4–6) on an DNA-oxoG using RTS, HeLa and Ogg1^−/− NE (kind gift of A Klungland). Quantitative transcription data (mean±s.d.) are derived from at least three independent experiments. (D) Repair of the damaged DNA templates as indicated at the top of the panel during the transcription reaction (lower panel). Templates (1464 bp), incubated in the presence of either RTS or HeLa NE, were next subjected to digestion by ApaLI. The presence of bands corresponding to 892 and 527 bp indicates digestion by ApaLI and thus repair of the lesion.

Figure 2

The bypass factor TFIIF. (A) Purification scheme from HeLa WCE. (B) Transcription, Coomassie staining, and TFIIF-Western blot using the fractions from the final heparin 5PW column. (C) Fr. 40–43 bypass activity towards several DNA lesions as indicated at the top of the panel. The quantification (mean±s.d.) was performed from at least three independent experiments. (D) TFIIF immunodepletion from the Pool of Fr.40–43 of the Heparin-5PW column (In), using either Ab-RAP74, an antibody raised against the larger subunit of TFIIF, or Ab-C, a control-antibody. The heavy (Hc) and light (Lc) chains are indicated. The proteins retained on the beads (B) as well as the ones in the flow through fraction (ΔIIF) were analyzed by SDS–PAGE (Coomassie staining, lanes 1–3) and Western blot (lanes 4–8) using the RAP74 and RAP30 antibodies. (E) These fractions (lanes 1–4) as well as the recombinant TFIIF (lanes 5–7) were further tested for their bypass activity in transcription. Quantitative data derived from at least three independent experiments are shown (mean±s.d., lower panels).

Figure 3

Elongation factor mediated specific bypass stimulation. (A) Scheme of transcription assay using immobilized DNA. After the first incubation (IncubI) with RTS and washes, the elongation factor such as TFIIF, CSB, Elongin or TFIIS was added for the second incubation (Incub II). (B) Western blot analysis of transcription components (In, lane 1), factors washed off (W, lane 2) or remaining bound (B, lane 3) to the immobilized damaged DNA. (C) Coomassie staining of purified recombinant CSB, Elongin and TFIIS. (D) Transcription on oxidative lesion containing DNA immobilized on magnetic beads in the presence of elongation factors (5 pmol) as indicated at the top of the panel. (E) Transcription as described above with a circular DNA template. Statistical analysis for (D) and (E) was performed by a pairwise comparison of elongation factor present versus negative control using a Student's t-test. Different degrees of significance were indicated as follows: ^*P<0.05, ^**P<0.01, ^***P<0.001. (F) TFIIS-stimulated transcript cleavage (TFIIS cleav.), sensitivity of transcription complexes halted on immobilized DNA-oxoG, DNA-Tg and DNA-CisPt. After the first incubation, when indicated, TFIIS with and without NTPs was added to the reaction.

Figure 4

RNA transcript sequence analysis. (A) ApaLI (10 U), digestion for 3 h at 37°C of the labelled RT–PCR-product from DNA-oxoG, DNA-Tg and DNA-OHdU templates following incubation with RTS. The uncut (330 nt) and ApaLI cut (169, 164, 163 and 159 nt) DNA fragments were analyzed on a 8% denaturing polyacrylamide gel. The ratios presented at the bottom of the figure shows the percentage of mutated transcript. (B) Transcript sequence analysis by RT–PCR product sequencing. During the transcription reaction, the indicated elongation factor was present. The position of the lesion is highlighted with a blue bar.