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

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

doi:10.3390/ijms22126589

Abstract

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.

Highlights

Introduction published maps and institutional affilEndonuclease XPG, belongs to the flap endonuclease (FEN) family of enzymes that cleave DNA at structure-specific sites [1,2]
We investigated the chromatin association of XPG during normal cell growth and after the inhibition of transcription, revealing a novel feature of XPG nuclear localization
Our results showed that this protein is accumulated at nuclear clusters when transcription is impaired both by RNA polymerase II (pol II) and topo I inhibitors

Summary

Introduction

Introduction published maps and institutional affilEndonuclease XPG, belongs to the flap endonuclease (FEN) family of enzymes that cleave DNA at structure-specific sites [1,2]. DNA incision activity by XPG is missing in a group of patients with xeroderma pigmentosum (XP) with mutations in the ERCC5 gene coding for XPG protein [6,7]. This inactivation leads to a cancer-prone phenotype, which is accompanied by severe deficiency in development, and by neurological disorder when XPG mutations are associated with Cockayne syndrome (CS), to produce a complex disease (XPG/CS) [8,9,10].

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Results

Conclusion