Abstract
Hereditary defects in the transcription-coupled nucleotide excision repair (TC-NER) pathway of damaged DNA cause severe neurodegenerative disease Cockayne syndrome (CS), however the origin and chemical nature of the underlying DNA damage had remained unknown. To find out, to which degree the structural properties of DNA lesions determine the extent of transcription arrest in human CS cells, we performed quantitative host cell reactivation analyses of expression vectors containing various synthetic adducts. We found that a single 3-(deoxyguanosin-N 2-yl)-2-acetylaminofluorene adduct (dG(N 2)-AAF) constitutes an unsurmountable obstacle to transcription in both CS-A and CS-B cells and is removed exclusively by the CSA- and CSB-dependent pathway. In contrast, contribution of the CS proteins to the removal of two other transcription-blocking DNA lesions – N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG(C8)-AAF) and cyclobutane thymine-thymine (TT) dimer – is only minor (TT dimer) or none (dG(C8)-AAF). The unique properties of dG(N 2)-AAF identify this adduct as a prototype for a new class of DNA lesions that escape the alternative global genome repair and could be critical for the CS pathogenesis.
Highlights
Cockayne syndrome (CS) is an incurable genetic disease characterized by severe neurological and developmental abnormalities, growth failure and pathological changes in multiple organs [1]
CS has been linked to a defect in the transcriptioncoupled nucleotide excision repair pathway (TC-NER), which is normally initiated by arrest of the elongating RNA polymerase II (RNAPII) complexes at bulky DNA adducts [2,3]
In a human CS-B cell line, we detected a potent inhibition of the gene expression by dG(N2)-AAF in the transcribed DNA strand of the enhanced green fluorescent protein (EGFP) gene, whereas the construct containing dG(C8)-AAF in the same position was expressed at the same level as control unmodified DNA (Figure 1)
Summary
Cockayne syndrome (CS) is an incurable genetic disease characterized by severe neurological and developmental abnormalities, growth failure and pathological changes in multiple organs [1]. CS has been linked to a defect in the transcriptioncoupled nucleotide excision repair pathway (TC-NER), which is normally initiated by arrest of the elongating RNA polymerase II (RNAPII) complexes at bulky DNA adducts [2,3]. Pathogenic mutations in CS were mapped to the genes CSB/ERCC6 (about 60% of cases) and CSA/ ERCC8 [5]. In response to UV damage, both gene products regulate (de-)ubiquitination reactions which are essential for displacement of the stalled RNAPII [6] – CSB as one of the initial sensors of the damage-arrested RNAPII [7] and CSA as an interaction partner of the CRL4 E3 ubiquitin ligase complex [8,9]. Since organs deep in the body are not exposed to UV, the nature and origins of DNA damage responsible for CS remain speculative [10,11]
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