We previously showed that treatment of cells with 6-TG and low doses of UVA causes oxidative DNA damage and that DNA 6-TG is a major target of oxidation. The same treatment also induces oxidation of DNA guanine to 8-oxoguanine (8-oxoG) (21
). Whilst cells are protected against the effects of DNA 8-oxoG by several efficient DNA repair pathways, we show here that 6-TG photoproducts are not excised from DNA. They induce a powerful, and largely irreversible, arrest of replication and transcription which triggers robust induction of the p53 DNA damage response (4
Conversion to thioguanine nucleotides and incorporation of 6-TG into DNA is a significant contributor to the clinical effects of thiopurines. 6-TG can also be incorporated into RNA (22
) although there appear to be fewer documented measurements of the extent of RNA substitution. In the cultured cells we used, RNA substitution by 6-TG is around 20-fold lower than that of DNA- and UVA-induced transcription inhibition in 6-TG treated cells is a consequence of photochemical DNA, rather than RNA, damage. Consistent with these findings, a single GSO3
in the transcribed DNA strand also arrests transcript elongation by RNAPII in an in vitro
assay. This easily identifiable product of oxidation of DNA 6-TG which is unable to form stable base pairs with canonical DNA bases (7
), is also a strong block to DNA replication in vitro
and it seems likely that an inability to direct an incoming NTP contributes to its effect on transcript elongation.
UVA introduces lesions in DNA containing 6-TG via the formation of ROS. In particular, singlet oxygen (1
) is generated in a Type II photochemical reaction. 1
is particularly hazardous because it damages not only DNA, but also proteins (8
). DNA transactions such as replication, repair, and transcription all involve proteins or protein complexes that interact intimately with DNA and might be particularly vulnerable to oxidation by 1
generated in DNA. The covalent oxidative crosslinking of PCNA subunits provides an example of this susceptibility (9
). Unlike PCNA, free RNA PolII does not form a closed toroidal structure. Nevertheless, the actively transcribing enzyme adopts a similar clamping strategy via
intimate and highly stable contacts with its DNA substrate and RNA product. 6-TG/UVA did not cause similar covalent crosslinking among RNAPII subunits. Instead, it provoked a rapid and extensive polyubiquitylation of Rpb1 and an as yet uncharacterized modification of the Rpb2 subunit, consistent with the presence of irreversible transcription-arresting photochemical DNA lesions (14
). In yeast, ubiquitylation of Rpb1 by the Def1 ubiquitylation factor is regarded as a strategy of last resort to clear irreversibly stalled RNAPII complexes by targeting them for degradation by the proteosome. In yeast, the Rpb1 subunit undergoes polyubiquitylation whereas Rpb2 apparently remains unmodified. In the human cells used in this study, we consistently observed modification of the Rpb2, but not the Rpb3 subunit. Whether this difference represents a slightly different strategy by which human cells deal with dangerous blocked transcription complexes is currently unclear. The observation provides additional evidence for the presence of obstructive DNA 6-TG photolesions in transcribed DNA of UVA irradiated cells.
Replication and transcription blocking DNA lesions are frequently substrates for NER, a versatile DNA repair system that removes a variety of bulky DNA adducts (23
). DNA GSO3
was not actively removed and persisted to the same extent in NER-proficient and defective cells. Defects in either of the two NER subpathways, global or transcription coupled (TCR) repair, are associated with UV sensitivity. The complete absence of NER—for example in xeroderma pigmentosum group A—confers extreme UVB sensitivity and susceptibility to sunlight-induced skin cancer. The selective TCR defects in Cockayne's syndrome (CS) Group A or B patients cause increased sunlight sensitivity which reflects the persistence of transcription-blocking UVB DNA photoproducts. TCR-proficient and CS cells responded to 6-TG/UVA by ubiquitylating the Rpb1 subunit of RNApolII, confirming the efficient detection of transcription-blocking DNA damage. The similar sensitivity of wild-type and CSB cells to 6-TG/UVA indicates that these transcription-inhibiting photoproducts are not subject to TCR. The XPA and CSB phenotypes are recapitulated in mouse models. Significantly, XPA and CSB mice are particularly susceptible to the induction of erythema/oedema by UVB (24
)—an observation that seems to connect the induction and persistence of transcription-blocking DNA lesions to these physiological end points. Csb−/−
mice are also susceptible to UVB-induced skin cancer. We have previously reported that the skin of patients taking azathioprine contains measurable DNA 6-TG (4
) and is hypersensitive to erythema induction by UVA but not UVB (26
). These observations suggest that the skin cells of these patients are particularly susceptible to the formation of transcription-blocking 6-TG/UVA photolesions. We note in this regard, that erythema in the form of sunburn, is a significant risk factor for the development of skin malignancies (27
). The link between sunlight and skin cancer is well established and mutations in critical tumor suppressor genes generally bear the hallmark of UVB exposure (28–30
). The composition of incident UV light and the deeper penetration of the longer UVA wavelengths means that vulnerable 6-TG-containing skin cells are exposed to up to 10 times more UVA than UVB. Investigation of the effects of UVA and DNA 6-TG in physiologically relevant cell types such as keratinocytes may now be warranted.
In summary, 6-TG/UVA induced damage to cellular DNA rapidly, and largely irreversibly, inhibits transcription. DNA 6-TG photoproducts, including the previously characterized GSO3, are potent blocks to transcript elongation by RNAPII in vitro. In agreement with this inhibitory effect on RNAPII, photoactivation of DNA 6-TG in vivo provokes the polyubiquitylation of RNAPII subunits that indicates persistent transcription blockage by DNA lesions. This RNAPII modification occurs in both NER competent and TCR-defective cells. The normal sensitivity of CSA and CSB cells to killing by 6-TG/UVA suggests that lethal 6-TG photoproducts have structural features that allow them to escape recognition and processing by excision repair. Persistent transcription-blocking DNA lesions induce erythema and are associated with the development of skin cancer. Our findings suggest that UVA may be a carcinogenic hazard for patients taking thiopurines.