Ubiquitin and TFIIH-stimulated DDB2 dissociation drives DNA damage handover in nucleotide excision repair

Abstract

DNA damage sensors DDB2 and XPC initiate global genome nucleotide excision repair (NER) to protect DNA from mutagenesis caused by helix-distorting lesions. XPC recognizes helical distortions by binding to unpaired ssDNA opposite DNA lesions. DDB2 binds to UV-induced lesions directly and facilitates efficient recognition by XPC. We show that not only lesion-binding but also timely DDB2 dissociation is required for DNA damage handover to XPC and swift progression of the multistep repair reaction. DNA-binding-induced DDB2 ubiquitylation and ensuing degradation regulate its homeostasis to prevent excessive lesion (re)binding. Additionally, damage handover from DDB2 to XPC coincides with the arrival of the TFIIH complex, which further promotes DDB2 dissociation and formation of a stable XPC-TFIIH damage verification complex. Our results reveal a reciprocal coordination between DNA damage recognition and verification within NER and illustrate that timely repair factor dissociation is vital for correct spatiotemporal control of a multistep repair process.

USP44 Stabilizes DDB2 to Facilitate Nucleotide Excision Repair and Prevent Tumors

“…Murine embryonic fibroblasts (MEFs) carrying combinations of hypomorphic or null alleles of Usp44 were generated and cultured as previously described (Zhang Y. et al, 2012). The human cell line VH10 expressing DDB2-GFP was generated and cultured as previously described (Pines et al, 2012;Ribeiro-Silva et al, 2020). The XPC deficient cell line XP4PA expressing XPC-GFP was generated and cultured as previously described.…”

“…The XPC deficient cell line XP4PA expressing XPC-GFP was generated and cultured as previously described. RNA interference was performed using smart pool reagents from Dharmacon according to manufacturer's instructions (Hoogstraten et al, 2008;Ribeiro-Silva et al, 2020). Cell fractionation was performed as described (Robu et al, 2013).…”

“…To better understand the impact of USP44 loss on DDB2 localization, we examined the kinetics of DDB2 accumulation on chromatin using live-cell fluorescent microscopy. As the level of DDB2 affects the NER reaction, these experiments were performed in human VH10 fibroblasts expressing DDB2-GFP at near endogenous levels (Pines et al, 2012;Ribeiro-Silva et al, 2020). Subnuclear DNA damage was induced using an UVC laser (266 nm) and the level of DDB2-GFP at the damage was monitored over time.…”

Chaperones for dancing on chromatin: Role of post‐translational modifications in dynamic damage detection hand‐offs during nucleotide excision repair

“…The following section details a new and exciting study that helps answer these questions and provides molecular insights into these well‐choreographed events. [ 38 ]…”

“…Despite our knowledge of ubiquitylation in NER, a clear mechanistic understanding was lacking. In this recent paper from Ribeiro‐Silva et al., at Erasmus University, [ 38 ] the authors used state‐of‐the‐art CRIPSR/Cas9 knockout and knock‐in approaches to follow tagged proteins that are expressed at endogenous levels. This is a key scientific advance as dysregulated levels of repair proteins can adversely affect specific steps in the repair cascade.…”