Position-dependent transcriptional impact and repair of DNA damage in the non-transcribed DNA strand of an active gene

Bulky DNA damage stalls RNA polymerase II when present in the transcribed strand of an active gene. As a response, cells initiate transcription-coupled nucleotide excision repair (TC-NER), which excises transcription-blocking damage. In contrast, lesions in the non-transcribed strand (NTS) are expected not to block transcription and are hence recognized by the other global-genome (GG-) NER subpathway through the XPC and DDB2 complexes. Preliminary findings in our lab demonstrated, unexpectedly, that UV-induced cyclobutane pyrimidine dimers (TT dimers) in the NTS can substantially suppress transcription when located in the 5′-untranslated region. We aimed to study this novel transcription-inhibiting effect (by damage in the NTS) by exploring whether lesions other than TT dimers can inhibit transcription, how such lesions are repaired, and which transcriptional components or phases can be potentially affected. Results showed that the bulky lesions of TT dimers and acetylaminofluorene (AAF) adducts in the NTS diminish transcription only when positioned between +21 and +24 base pairs downstream of the transcription start site. We propose a model in which such lesions interact with transcription factors that associate with the NTS within this narrow window, potentially components of the preinitiation complex or factors involved in the transition from initiation to elongation. Repair analysis revealed that TC-NER can repair transcription-inhibiting lesions in the NTS, albeit with low efficiency. It also revealed that GG-NER exhibits lesion-specific activity: it fully repairs C8-AAF adducts in a DDB2-independent manner, partially repairs TT dimers, but fails to repair N2-AAF. These results align with the understanding that NER sub-pathways do not respond uniformly to a predefined set of bulky or helix-distorting lesions. Rather, GG-NER and TC-NER exhibit lesion-specific responses, based on the structural features and biological consequences of each single modification.