Transcription-coupled repair and apoptosis provide specific protection against transcription-associated mutagenesis by ultraviolet light

Abstract

Recent data reveal that gene transcription affects genome stability in mammalian cells. For example, transcription of DNA that is damaged by the most prevalent exogenous genotoxin, UV light, induces nucleotide substitutions and chromosomal instability, collectively called UV-induced transcription-associated mutations (UV-TAM ). An important class of UV-TAM consists of nucleotide transitions that are caused by deamination of cytosine-containing photolesions to uracil, presumably occurring at stalled transcription complexes. Transcription-associated deletions and recombinational events after UV exposure may be triggered by collisions of replication forks with stalled transcription complexes. In this Point-of-View, we propose that mammalian cells possess two tailored mechanisms to prevent UV-TAM in dermal stem cells. First, the transcription-coupled nucleotide excision repair (TCR ) pathway removes lesions at transcribed DNA strands, forming the primary barrier against the mutagenic consequences of transcription at a damaged template. Second, when TCR is absent or when the capacity of TCR is exceeded, persistently stalled transcription complexes induce apoptosis, averting the generation of mutant cells following replication. We hypothesize that TCR and the apoptotic response in conjunction reduce the risk of skin carcinogenesis.

Transcription-Associated Mutagenesis at UV-Photolesions

Transcription is quintessential for life. Although at least 50% of the mammalian genome is transcribed, frequently into regulatory RNA molecules,¹ only a minor fraction of the genome consists of protein-encoding genes that can be transcribed by a multiprotein complex that includes RNA polymerase II. In the last decades, evidence has accumulated that there is also a downside to transcription. Research conducted mainly in bacteria and lower eukaryotes has demonstrated that a high level of transcription at undamaged templates induces genome instability, including nucleotide substitution mutations, deletions and chromosome rearrangements.^2,3 In mammals these classes of mutations are associated with the oncogenic derailment of stem cells. Also transcription at damaged templates is associated with genomic instability. Ultraviolet (UV) light is a prevalent environmental mutagen and carcinogen. We have recently found in mouse embryonic stem (ES) cells that UV light-induced lesions at the template strand for transcription in a reporter gene become much more mutagenic when the gene is transcribed.⁴ Two novel classes of UV-induced transcription-associated mutations (UV-TAM) were found. The first class was comprised of nucleotide substitutions, by a pathway dubbed UV-induced transcription-associated point mutagenesis (UV-TAP). Specifically, frequencies of CC to TT and C to T transition mutations, derived from photolesions residing at the template strand for transcription, were increased when the gene was transcribed during and after UV exposure of the cells.⁵ We have found that dicytosines, embedded within a cyclobutane pyrimidine dimer (CPD) photolesion, are prone to deamination to UU when the template is transcribed.⁵ During replication, these deoxyuridine residues within a CPD photolesion direct the incorporation of deoxyadenosine, leading to the observed CC to TT transition mutations. Although these were not separately investigated, transcription-associated single C to T transitions are most probably generated by the same mechanism. Based on these and previous⁶ studies we infer that it is the prolonged presence of cytosine-containing CPDs in single-stranded DNA at the stalled transcription complex that predisposes them to spontaneous deamination. Since particularly cytosines that reside in photolesions are prone to deamination,⁶ this class of TAM is probably restricted to UV-induced DNA damage. Supporting this hypothesis, Benzo(a)pyrene diolepoxyde (BPDE) induces transcription-associated transversion mutations originating from lesions at the non-transcribed strand for transcription (Hendriks et al., in preparation),⁴ in sharp contrast to UV that induces transcription-associated transitions at the transcribed strand.

In addition to inducing nucleotide transitions, UV exposure at an active reporter gene induced transcriptionassociated deletions (UV-TAD) that are most likely caused by error-prone end joining of double stranded DNA breaks (DSBs). These DSBs may mechanistically be related to those that underlie transcription-associated recombination (TAR) in mammalian cells, in the absence of template damage⁷ or at UV-damaged templates.⁸ At undamaged templates, DNA-RNA hybrids (R-loops) that persist behind the transcribing RNA polymerase II, are involved in the induction of DSBs.⁹ The dependence of TAR on replication indicates that a replication fork that encounters an R-loop may result in the collapse of the fork and the generation of a DSB.⁷ We hypothesize that, when RNA polymerase II remains stalled at photolesions within the template.¹⁰ R loops persist, which increases their chance of collision with replication forks. Also BPDE induces TAD in ES cells, suggesting that TAD may be triggered by any lesion that arrests transcription (manuscript in preparation).

Transcription-Coupled Nucleotide Excision Repair Provides Specific Protection Against UV-TAM

DNA lesions that arrest transcription can be removed from the template DNA by the transcription-coupled sub-pathway of nucleotide excision repair (TCR).¹¹ TCR is initiated by stalling of the transcription machinery at a DNA lesion, resulting in the recruitment of nucleotide excision repair (NER) factors that remove the lesion from the DNA. In this fashion, the recovery of transcription after exposure to DNA-damaging agents is ensured, and this generally is considered to be the primary function of TCR (Fig. 1A).¹¹ However, the specific characteristics of UV-TAM provide an additional raison d'être for TCR (Fig. 1B). Most DNA-damaging agents induce nucleotide substitution mutagenesis via error-prone translesion synthesis opposite the template lesion during DNA replication. In contrast, UV-TAM is determined by deamination during transcription and by subsequent error-free (but mutagenic) translesion synthesis, presumably by DNA polymerase η.¹² Since they are extremely mutagenic, cytosinecontaining CPD photolesions at a stalled transcription complex or their deaminated derivatives, should efficiently be repaired. CPDs are a relatively poor substrate for global-genome NER.^13,14 This puts the burden of preventing UV-induced TAP on TCR, which efficiently removes CPDs from the transcriptional template. In conclusion, TCR forms the major barrier against the deamination-dependent mutagenicity of cytosine-containing CPDs, in addition to its roles in transcription recovery and in preventing UV-TAR and presumably TAD.^4,5,8

Figure 1.

Model for the roles of TCR and transcription arrest-associated apoptosis in protection against UV-TAM. (A) TCR is initiated by stalling of the elongating RNA polymerase and results in removal of the transcription-blocking photolesion, thereby allowing transcription recovery. In case the load of photolesions exceeds the capacity of TCR or when TCR is impaired, arrested transcription complexes induce apoptosis when the cell enters S phase. (B) Persistent stalling of the transcription complex at a deoxycytidine-containing-CPD at the template strand results in its increased spontaneous deamination frequency. In case the lesion is not timely removed by TCR, or when the cell escapes from apoptosis, it will result in a C to T transition mutation during replication. Alternatively, persistent stalling of the transcription complex at a photolesion may lead to collapse of an approaching replication fork, resulting in DSBs and genome instability.

Our finding of UV-TAP and its prevention by TCR explains previous data from others. For instance, most UV-induced C to T transitions at the Hprt locus in TCR-deficient hamster and human cells are derived from photolesions at the transcribed strand.¹⁵ Cells that are deficient for Cockayne Syndrome B (CSB) protein, a key factor in TCR, show a strong bias in mutations induced by photolesions at the template strand for transcription and an excessive apoptotic response when exposed to UV light.¹⁶

Transcription Arrest, Apoptosis and TAM

TCR-deficient epidermal cells in situ display high levels of apoptosis upon the infliction of DNA damage, indicating that the inability to remove lesions at the template strand for transcription efficiently initiates DNA damage responses.¹⁹ Indeed, persistent stalling of the elongating transcription complex induces strong p53 activation.²⁰ It was hypothesized that the transcription arrest-induced apoptosis serves as a general DNA damage sensor, resulting in the elimination of cells that contain high levels of DNA damage (Fig. 1A). This would provide additional protection against the possible mutagenic and carcinogenic consequences of unrepaired genomic DNA damage in mammals.^16,21,22 Although attractive, such a hypothesis contains a paradoxical element. As argued above, mutations usually are generated during error-prone replication of damaged DNA, rather than during transcription. This suggests that only replicating cells are at risk for mutagenesis upon the infliction of DNA damage and need to initiate apoptosis when the damage load entails a too high risk for losing genome integrity. In agreement, when replication is arrested at lesions, DNA damage responses are efficiently induced.^20,23 What would be the added benefit of the induction of DNA damage responses by arrested transcription complexes in non-replicating cells, when genome stability is threatened only during replication? The finding that UV-TAP is determined at the level of (arrested) transcription, rather than during replication, may provide an answer to this conundrum. If the transcriptionarresting UV lesions are not repaired efficiently by TCR, deamination of cytosine-containing CPDs may occur and this will inevitably result in the induction of transition mutations when the cell would subsequently enter S phase. Similarly, the persistence of R-loops at transcription complexes, arrested at lesions within the template, entails a very high risk of replication fork collapse, genomic rearrangements and deletions. To avoid such detrimental TAM, transcription arrests sensitize the cells to apoptosis, so that if the cells enter S phase, the apoptotic pathway is effectuated. In support, the efficient induction of apoptosis by arrested transcription complexes requires replication.²¹ Summarizing, the induction of apoptosis by transcription-arresting lesions depends on the integration of DNA damage responses that are induced by both transcription arrests and replication.

Our and others' data^4,5,8 indicate that TCR provides a rather moderate level of protection against UV-TAM. UV doses that cause similar frequencies of UV-TAM in NER-proficient and NER-deficient ES cells, reflecting similar numbers of persisting lesions, induce very similar cell death.^4,5 This finding indicates the presence of a threshold level of lesions at the template strands for transcription above which transcriptional stalling will induce apoptosis, when cells enter S phase. We hypothesize that this strong apoptotic response will eliminate cells with an excessive risk of UV-TAM and, by implication, prevents associated (skin) carcinogenesis.

Despite their TCR deficiency, CSBdeficient humans develop no UV-induced skin cancer.¹⁷ These patients are proficient for the global genome NER that might remove a fraction of the mutagenic CPD lesions from transcribed templates in the absence of TCR, thereby suppressing carcinogenesis, as was recently found for CSB-deficient mice.¹⁸ Nevertheless, the remarkable lack of skin cancer supports the idea that the efficient induction of apoptosis by arrested transcription complexes, despite the presence of TAP, provides protection against oncogenic transformation of the cell.

Conclusions and Perspective

The discovery of two novel classes of UV-TAM may provide a new rationale for the interacting roles of TCR and of transcription arrest-induced apoptosis in responses to UV light-induced DNA damage. We and others³ hypothesize that these responses are crucial in preventing the emergence of mutant cell clones. This is of particular relevance for transcribed growth-controlling genes in mammalian dermal stem cells as they prevent the oncogenic derailment of these cells. Further research using defined mouse models for UV-TAM should bear this out.