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 0 -cyclo-2 0 -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 0 ) 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 helixdistorting DNA lesions that are substrates for nucleotide excision repair, resulting in transcriptional mutagenesis.

Mammalian Transcription-Coupled Excision Repair

“…However, this is probably a rare event as the presence of a CPD in the active site of RNAPII has been shown to strongly disfavor forward translocation of RNAPII ). Under certain conditions and likely with low frequency, bypass of helix distorting lesions such as CPDs and cyclo-dA may occur in yeast (Walmacq et al 2012) and in NER deficient human cells (Marietta and Brooks 2007). Yeast RNAPII was shown to bypass CPDs via an intrinsic ability to perform errorfree translesion synthesis, whereas bypass of bulky lesions in human XP-A cells resulted not only in transcription mutagenesis but also in nonmutant transcripts.…”

“…Stalled transcription forms an acute problem for cellular homeostasis by depriving cells of vital messages. In addition, DNA lesions in the transcribed strand may induce mutant transcripts as a consequence of transcriptional bypass over DNA lesions (Doetsch 2002;Marietta and Brooks 2007). A sophisticated subpathway of NER has been evolved to specifically resolve transcription complexes stalled at DNA lesions, thus allowing restoration of transcription and successful production of essential transcripts.…”

“…Based on experimental observations, several models have been proposed for the way cells respond when the progression of an actively transcribed RNAPII is blocked by DNA lesions and for the fate of the polymerase in cases of faulty repair. These involve stalling of RNAPII and its dislocation from the damaged chromatin, either by reverse translocation (backtracking) leading to RNAPII arrest or by dissociation from the chromatin and subsequent degradation of the polymerase, as well as lesion bypass by RNAPII Marietta and Brooks 2007;Fousteri and Mullenders 2008;Cheung and Cramer 2011;Walmacq et al 2012;Wilson et al 2012). RNAPII has been shown to stall at CPDs for at least 20 h in vitro (Selby et al 1997) and for more than 48 hours in vivo in Csb-deficient mouse cells after UV-C irradiation (Garinis et al 2009).…”

The tomato DDI2, a PCNA ortholog, associating with DDB1-CUL4 complex is required for UV-damaged DNA repair and plant tolerance to UV stress

“…As sessile organisms, plants are inevitably affected by UV radiation and other environmental stresses. UV light can penetrate cells and cause DNA damage; in particular cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts [(6-4)PPs], that blocks or renders inaccurate replication and transcription; and leads to the mutagenic consequences [2]. For surviving, plants have evolved a number of adaptive strategies to overcome the stresses; such as physiological adaptation like biosynthesis of UV-absorbing compounds [3]; photoreactivation [1]; nucleotide excision repair (NER) [4] and translesion synthesis (TLS) [5].…”

8-Oxoguanine-mediated transcriptional mutagenesis causes Ras activation in mammalian cells

“…The small number of in vivo studies conducted to date have relied on luciferase reporter assays in bacterial cells to gauge TM caused by uracil or 8OG (13,14). Interestingly, a recent study of transcriptional encounters with 8,5Ј-cyclo-2Ј-deoxyadenosine and cyclobutane pyrimidine dimers in mammalian cells indicated that even bulky lesions that normally pose a significant block to transcription can be occasionally bypassed with mutagenic effects (15).…”