Cryo-EM structure of TFIIH/Rad4\u2013Rad23\u2013Rad33 in damaged DNA opening in nucleotide excision repair

Trevor Van Eeuwen,Tingting Zhao,Yoonjung Shim,Benjamin A Garcia,Jung-Hyun Min,Craig D Kaplan,Hee Jong Kim,Kenji Murakami,Shrabani Basu

doi:10.1038/s41467-021-23684-x

Trevor Van Eeuwen, Tingting Zhao + Show 7 more

Open Access

https://doi.org/10.1038/s41467-021-23684-x

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Abstract

The versatile nucleotide excision repair (NER) pathway initiates as the XPC–RAD23B–CETN2 complex first recognizes DNA lesions from the genomic DNA and recruits the general transcription factor complex, TFIIH, for subsequent lesion verification. Here, we present a cryo-EM structure of an NER initiation complex containing Rad4–Rad23-Rad33 (yeast homologue of XPC–RAD23B–CETN2) and 7-subunit coreTFIIH assembled on a carcinogen-DNA adduct lesion at 3.9–9.2 Å resolution. A ~30-bp DNA duplex could be mapped as it straddles between Rad4 and the Ssl2 (XPB) subunit of TFIIH on the 3' and 5' side of the lesion, respectively. The simultaneous binding with Rad4 and TFIIH was permitted by an unwinding of DNA at the lesion. Translocation coupled with torque generation by Ssl2 and Rad4 would extend the DNA unwinding at the lesion and deliver the damaged strand to Rad3 (XPD) in an open form suitable for subsequent lesion scanning and verification.

Highlights

The versatile nucleotide excision repair (NER) pathway initiates as the XPC–RAD23B–centrin 2 (CETN2) complex first recognizes DNA lesions from the genomic DNA and recruits the general transcription factor complex, TFIIH, for subsequent lesion verification
To assemble a TFIIH/Rad4–Rad23–Rad33/DNA complex suitable for structural study, the 7-subunit coreTFIIH was isolated from yeast as previously published[46] with slight modifications
Our model suggests that this is due to the role of C-XPC (Rad4) in binding to CETN2 (Rad33) which in turn directly interacts with the C-terminal helix of XPB (Ssl2)

Summary

Introduction

The versatile nucleotide excision repair (NER) pathway initiates as the XPC–RAD23B–CETN2 complex first recognizes DNA lesions from the genomic DNA and recruits the general transcription factor complex, TFIIH, for subsequent lesion verification. We present a cryo-EM structure of an NER initiation complex containing Rad4–Rad23-Rad[33] (yeast homologue of XPC–RAD23B–CETN2) and 7-subunit coreTFIIH assembled on a carcinogenDNA adduct lesion at 3.9–9.2 Å resolution. Rad[4] interacts with DNA through its four consecutive domains: the transglutaminase domain (TGD) and the β-hairpin domain 1 (BHD1) bind to a ~11-bp undamaged segment of the DNA duplex, whereas BHD2 and BHD3 together bind to a 4-bp segment harboring the damage This binding results in an ‘open’ DNA conformation where the damaged site is locally unwound and two damagecontaining nucleotide pairs are flipped out of the DNA duplex, while a β-hairpin from BHD3 plugs into the gap in the duplex to stabilize such conformation[25,26]. Further kinetic and single-molecule studies have revealed that Rad[4] performs fast diffusional search while probing the DNA structural integrity that involves untwisting[24,25,26]

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