Cryo-EM of NHEJ supercomplexes provides insights into DNA repair

Amanda K Chaplin,Steven W Hardwick,Antonia Kefala Stavridi,Christopher J Buehl,Noah J Goff,Virginie Ropars,Shikang Liang,Taiana Maia De Oliveira,Dimitri Y Chirgadze,Katheryn Meek,Jean-Baptiste Charbonnier,Tom L Blundell

doi:10.1016/j.molcel.2021.07.005

Abstract

SUMMARYNon-homologous end joining (NHEJ) is one of two critical mechanisms utilized in humans to repair DNA double-strand breaks (DSBs). Unrepaired or incorrect repair of DSBs can lead to apoptosis or cancer. NHEJ involves several proteins, including the Ku70/80 heterodimer, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), X-ray cross-complementing protein 4 (XRCC4), XRCC4-like factor (XLF), and ligase IV. These core proteins bind DSBs and ligate the damaged DNA ends. However, details of the structural assembly of these proteins remain unclear. Here, we present cryo-EM structures of NHEJ supercomplexes that are composed of these core proteins and DNA, revealing the detailed structural architecture of this assembly. We describe monomeric and dimeric forms of this supercomplex and also propose the existence of alternate dimeric forms of long-range synaptic complexes. Finally, we show that mutational disruption of several structural features within these NHEJ complexes negatively affects DNA repair.

Highlights

In all kingdoms of life, the ability to repair DNA damage is essential
BRCT1 of ligase IV (LigIV) interacts with Ku70/80 The major interaction site between DNA-PK and LX4 involves the BRCT1 domain of LigIV and Ku70/80
The crystal structure of X-ray cross-complementing protein 4 (XRCC4) with the BRCT tandem repeats of LigIV can be docked into this additional density of our cryoelectron microscopy (cryo-EM) map with a minor rotation of the BRCT1 domain relative to XRCC4 compared with the X-ray crystal structure (Figure S2F)

Summary

Introduction

In all kingdoms of life, the ability to repair DNA damage is essential. Non-homologous end joining (NHEJ) is nearly ubiquitous as a DNA repair mechanism and is one of the two main mechanisms utilized to repair DNA double-strand breaks (DSBs) in humans (Harper and Elledge, 2007). In NHEJ, DSBs are first recognized by the Ku70/80 heterodimer, which subsequently recruits the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a large protein kinase belonging to the phosphoinositide-3-kinase-related kinase (PIKK) family (Hartley et al, 1995). We recently improved the resolution of the DNA-PK holoenzyme to 3.5-Aresolution following density modification and revealed a dimer of DNA-PK mediated via the C terminus of Ku80, which had not been reported previously (Chaplin et al, 2021). This dimer provided new structural insights into the NHEJ mechanism, and we hypothesized its ability to act as a central stage for further recruitment and regulation of downstream NHEJ proteins

Results

Discussion

Conclusion