Transcriptional Stress Induces Chromatin Relocation of the Nucleotide Excision Repair Factor XPG.

Claudia Scalera, Giulio Ticli, Ilaria Dutto, Ornella Cazzalini, Lucia A Stivala, Ennio Prosperi

International journal of molecular sciences 2021 Jun 19

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Endonuclease XPG participates in nucleotide excision repair (NER), in basal transcription, and in the processing of RNA/DNA hybrids (R-loops): the malfunction of these processes may cause genome instability. Here, we investigate the chromatin association of XPG during basal transcription and after transcriptional stress. The inhibition of RNA polymerase II with 5,6-dichloro-l-β-D-ribofuranosyl benzimidazole (DRB), or actinomycin D (AD), and of topoisomerase I with camptothecin (CPT) resulted in an increase in chromatin-bound XPG, with concomitant relocation by forming nuclear clusters. The cotranscriptional activators p300 and CREB-binding protein (CREBBP), endowed with lysine acetyl transferase (KAT) activity, interact with and acetylate XPG. Depletion of both KATs by RNA interference, or chemical inhibition with C646, significantly reduced XPG acetylation. However, the loss of KAT activity also resulted in increased chromatin association and the relocation of XPG, indicating that these processes were induced by transcriptional stress and not by reduced acetylation. Transcription inhibitors, including C646, triggered the R-loop formation and phosphorylation of histone H2AX (γ-H2AX). Proximity ligation assay (PLA) showed that XPG colocalized with R-loops, indicating the recruitment of the protein to these structures. These results suggest that transcriptional stress-induced XPG relocation may represent recruitment to sites of R-loop processing.

Citation

Claudia Scalera, Giulio Ticli, Ilaria Dutto, Ornella Cazzalini, Lucia A Stivala, Ennio Prosperi. Transcriptional Stress Induces Chromatin Relocation of the Nucleotide Excision Repair Factor XPG. International journal of molecular sciences. 2021 Jun 19;22(12)