Malondialdehyde adducts in DNA arrest transcription by T7 RNA polymerase and mammalian RNA polymerase II

Abstract

Malondialdehyde, a genotoxic byproduct of lipid peroxidation, reacts with guanine in DNA to form pyrimido[1,2-alpha]purin-10(3H)one (M(1)dG), the first endogenous DNA lesion found to be a target of nucleotide excision repair enzymes. A subpathway of nucleotide excision repair, transcription-coupled repair, is thought to occur when RNA polymerase (RNAP) is arrested at damage in transcribed DNA strands and might function for efficient removal of M(1)dG in active genes. Results presented here show that M(1)dG and its stable, exocyclic analog 1,N(2)-propanodeoxyguanine (PdG), arrest translocation of T7 RNAP and mammalian RNAPII when located in the transcribed strand of a DNA template. M(1)dG paired with thymine is exocyclic and poses a stronger block to transcription than the acyclic N(2)-(3-oxo-1-propenyl)-dG, formed upon cytosine-catalyzed opening of M(1)dG in duplex DNA. PdG is a complete block to RNAPII regardless of base pairing. The elongation factor TFIIS (SII) induces reversal and RNA transcript cleavage by RNAPII arrested at PdG. Thus, arrested RNAPII complexes may be stable at M(1)dG in cells and may resume transcription once the offending adduct is removed. The conclusion from this work is that malondialdehyde adducts in the transcribed strand of expressed genes are strong blocks to RNAPs and are targets for cellular transcription-coupled repair. If so, then M(1)dG, already known to be highly mutagenic in human cells, also may contribute to apoptosis in the developing tissues of individuals with Cockayne's syndrome, a hereditary disorder characterized by transcription-coupled repair deficiency.

Fig. 1.

Formation of M₁dG and the structures of M₁dG and PdG.

Fig. 2.

DNA substrates for RNAP transcription. The linear Xts and Xnts DNA templates contain dG, M₁dG, or PdG (X) either in the transcribed or nontranscribed strands, respectively, downstream of promoters for T7RNAP (T7P) and RNAPII (AdMLP) transcription. Numbers in parentheses indicate positions in the nontranscribed strand of the DNA plasmid construct. Transcription starts at each promoter (+1) and proceeds in the direction of the bent arrows. Transcripts from the T7 promoter incorporate [α-³²P]GTP and from AdMLP incorporate [α-³²P]CTP within the labeling cassette. Stalled complexes restart at the end of the cassette (+5 from the T7 promoter and +15 from AdMLP). Transcript lengths expected from runoff (RO) or from arrest at the X are depicted by lines below each template. The XhoI-BamHI 34-bp sequence inserted in the plasmid construct as described in Materials and Methods is shown with the 18-base primer sequence in an enlarged type size.

Fig. 3.

T7RNAP elongation is arrested by M₁dG and PdG in the transcribed strand. The ³²P-labeled transcripts from T7RNAP elongation on DNA templates with the adduct position paired with C (A) or T (B) are shown. In each panel, lanes 1, 3, and 5 contain transcripts from Xts (T) and lanes 2, 4, and 6 transcripts from Xnts (N) containing dG, M₁dG, and PdG, respectively. A 10-base DNA ladder is resolved in lane L. RO, runoff transcripts; P, pause site transcripts; X, adduct-arrested transcripts; MS, minor arrest site transcripts.

Fig. 4.

T7RNAP transcription time course for arrest at C-paired adducts in the transcribed strand. (A) T7RNAP arrest at M₁dG (lanes 1-7) or PdG (lanes 8-14) paired with C in the Xts template was observed for 60 min after the initiation of elongation by NTP addition. Transcripts after a 5-min elongation on the template with dG are in lane 15. RO, runoff transcripts; X, adduct-arrested transcripts. (B) A graph of the % of transcripts arrested at X (•, M₁dG arrest; ▪, PdG arrest) and the % of full-length transcripts (○, RO past M₁dG; □, RO past PdG) for each template over the T7RNAP elongation time course.

Fig. 5.

T7RNAP transcription time course for arrest at T-paired adducts in the transcribed strand. (A) T7RNAP arrest at M₁dG (lanes 1-7) or PdG (lanes 8-14) paired with C in the Xts template was observed for 60 min after the initiation of elongation by NTP addition. Transcripts after a 5-min elongation on the template with dG are in lane 15. RO, runoff transcripts; X, adduct-arrested transcripts. (B) A graph of the % of transcripts arrested at X (•, M₁dG arrest; ▪, PdG arrest) and the % of full-length transcripts (○, RO past M₁dG; □, RO past PdG) for each template over the T7RNAP elongation time course.

Fig. 6.

M₁dG and PdG block transcript elongation by RNAPII. ³²P-labeled transcripts from RNAPII elongation on the Xts (TS) with X opposite C (A, lanes 1-3) or opposite T (B, lanes 1-3) are shown along with transcripts from RNAPII elongation on Xnts (NTS) opposite T (B, lanes 4-6). The base at X is indicated above each lane (G, dG; M, M₁dG; P, for PdG). RO, runoff transcripts; X, adduct-arrested transcripts.

Fig. 7.

RNAPII complexes arrested at PdG respond to SII. Transcripts arrested at PdG opposite a C in the Xts template (X) were cleaved after SII addition (X_SII, lanes 2 and 3).