Transcriptional bypass of bulky DNA lesions causes new mutant RNA transcripts in human cells

Abstract

Here, we characterize the mutant transcripts resulting from bypass of an 8,5'-cyclo-2'-deoxyadenosine (cyclo-dA) or cyclobutane pyrimidine dimer (CPD) by human RNA polymerase II (Pol II) in vivo. With the cyclo-dA lesion, we observed two new types of mutant transcripts. In the first type, the polymerase inserted uridine opposite the lesion and then misincorporated adenosine opposite the template deoxyadenosine downstream (5') of the lesion. The second type contained deletions of 7, 13 or 21 nucleotides (nt) after uridine incorporation opposite the lesion. The frequency of the different types of transcript from the cyclo-dA lesion in mutant human cell lines suggests that the Cockayne syndrome B protein affects the probability of deletion transcript formation. With the CPD-containing construct, we also detected rare transcripts containing 12 nt deletions. These results indicate that RNA pol II in living human cells can bypass helix-distorting DNA lesions that are substrates for nucleotide excision repair, resulting in transcriptional mutagenesis.

Figure 1

An experimental strategy for detecting lesion bypass by RNA. (A) Structures of the DNA lesions cyclo-dA and CPD investigated in this study. (B) Strategy for analysis of transcriptional mutagenesis by cyclo-dA or a CPD in vivo. Double-stranded DNA constructs containing site-specific DNA lesions are prepared and transfected into NER-deficient cells. The V on the construct represents the location of the intron. A indicates the location of the cyclo-dA and T-T indicates the location of the CPD on the transcribed strand of the luciferase gene. The spliced RNAs (Δ in the messenger RNA represents the splice site) containing mutations resulting from bypass of the lesion (X) in the cell are amplified by RT-PCR, cloned into plasmids, screened for mutations by MfeI digestion and the mutants are sequenced. (C) RT–PCR analysis of RNA from cells transfected with cyclo-dA (lane 1), CPD (lane 2) or lesion-free constructs (lane 3). No band was detected in the absence of input RNA (lane 4). The band is the expected size of the PCR product derived from spliced RNA (357 bp), whereas no band is detectable at the position where amplified plasmid DNA (489 bp) would migrate. The PCR conditions used are not suitable for quantitative comparisons. CPD, cyclobutane pyrimidine dimer; cyclo-dA, 8,5′-cyclo-2′-deoxyadenosine; NER, nucleotide excision repair; RT–PCR, reverse transcription–PCR.

Figure 2

An 8,5′-cyclo-2′-deoxyadenosine lesion stimulates a novel type of misincorporation event when transcribed by polymerase II. (A) The sequences of the 5′A and WT transcripts are indicated above the double-stranded DNA construct, showing the location of the cyclo-dA lesion (A). (B) Sequence electropherogram of the cDNAs corresponding to the WT or 5′A transcripts. (C) The predicted open amino-acid sequences of the WT and 5′A mutant transcripts are the same. 5′A, misincorporation of adenosine opposite the template dA 5′ to the lesion; cyclo-dA, 8,5′-cyclo-2′-deoxyadenosine; WT, wild type.

Figure 3

Transcripts containing stepwise multiple nucleotide deletions resulting from bypass of an 8,5′-cyclo-2′-deoxyadenosine lesion or a cyclobutane pyrimidine dimer lesion. (A) The sequences of the MND transcripts are indicated above the double-stranded DNA construct, showing the location of the cyclo-dA lesion (red A). The underlined nucleotides represent potential template nucleotides for resuming transcription (see Fig 4). Sequence electropherograms of cDNAs corresponding to the –7, −13 and −21 nucleotide (nt) deletion transcripts are below. (B) The sequence of the −12 nt deletion transcript obtained from cells transfected with a CPD is indicated above the double-stranded DNA construct showing the location of the CPD (red T-T). The underlined nucleotides represent potential template nucleotides for resuming transcription. A sequence electropherogram of the −12 nt cDNA is shown below. CPD, cyclobutane pyrimidine dimer; cyclo-dA, 8,5′-cyclo-2′-deoxyadenosine; MND, multiple nucleotide deletions.

Figure 4

A speculative model for multiple nucleotide deletion transcript formation. The violet rounded rectangle represents polymerase II and CM indicates the clamp module (Gnatt et al, 2001). The RNA transcript is shown in yellow, template DNA strand in white, non-template strand in grey and the portion of the template strand that is ‘skipped' in blue. The A represents the cyclo-dA lesion and the blue A represents the downstream template dA residue used to reinitiate transcription (underlined in Fig 3). The proposed tethering protein is not shown. After incorporating U opposite the cyclo-dA lesion and stalling (A), the CM partially opens, allowing dissociation of the hybrid, but with retention of the nascent RNA (see Liu et al, 1993; B). Downstream DNA is pulled into the polymerase past the RNA transcript and extruded upstream by a scrunching mechanism (C). The CM closes on the ‘hybrid' and the extruded DNA rewinds, allowing transcription to continue (D). As discussed in the text, other factors in addition to the polymerase are likely to be necessary, but are not shown for clarity. Cyclo-dA, 8,5′-cyclo-2′-deoxyadenosine; U, uridine.