A senataxin-associated exonuclease SAN1 is required for resistance to DNA interstrand cross-links

Alex M Andrews,Ian G Macara,Heather J Mccartney,Alan D D’Andrea,Tim M Errington

doi:10.1038/s41467-018-05008-8

Abstract

Interstrand DNA cross-links (ICLs) block both replication and transcription, and are commonly repaired by the Fanconi anemia (FA) pathway. However, FA-independent repair mechanisms of ICLs remain poorly understood. Here we report a previously uncharacterized protein, SAN1, as a 5′ exonuclease that acts independently of the FA pathway in response to ICLs. Deletion of SAN1 in HeLa cells and mouse embryonic fibroblasts causes sensitivity to ICLs, which is prevented by re-expression of wild type but not nuclease-dead SAN1. SAN1 deletion causes DNA damage and radial chromosome formation following treatment with Mitomycin C, phenocopying defects in the FA pathway. However, SAN1 deletion is not epistatic with FANCD2, a core FA pathway component. Unexpectedly, SAN1 binds to Senataxin (SETX), an RNA/DNA helicase that resolves R-loops. SAN1-SETX binding is increased by ICLs, and is required to prevent cross-link sensitivity. We propose that SAN1 functions with SETX in a pathway necessary for resistance to ICLs.

Highlights

Interstrand DNA cross-links (ICLs) block both replication and transcription, and are commonly repaired by the Fanconi anemia (FA) pathway
Many components of these pathways are conserved between yeast and higher organisms, animals have evolved an additional network of >20 proteins specialized for ICL repair, called the Fanconi anemia (FA) pathway[1,2]
R-loops can be resolved by an endonuclease, RNase H, or by an RNA/DNA helicase, senataxin (SETX), and if they persist can be aberrantly processed into double stranded breaks (DSBs) by the nucleotide excision repair (NER) endonucleases XPF and XPG14

Summary

Introduction

Interstrand DNA cross-links (ICLs) block both replication and transcription, and are commonly repaired by the Fanconi anemia (FA) pathway. We identified an uncharacterized protein that contains an N-terminal domain closely related to the FEN1 family of structure-specific nucleases This protein is not a known component of any DNA repair complex, but we report that it is a 5′-exonuclease for single-stranded (ss) DNA, and is required for the cellular response to ICLs. Disruption of the gene for this nuclease, Fam120b, has no effect on cell cycle but increases sensitivity to cross-linking agents, and results in DNA damage and radial chromosome formation in the presence of Mitomycin C (MMC). Disruption of the gene for this nuclease, Fam120b, has no effect on cell cycle but increases sensitivity to cross-linking agents, and results in DNA damage and radial chromosome formation in the presence of Mitomycin C (MMC) It is not epistatic with components of the FA pathway including FANCD2 and XPF. It is the first nuclease to be identified in the cellular response to ICLs since the discovery of FAN1, and will provide an important gateway to understand FA-independent mechanisms of DNA repair required for the resolution of ICLs

Methods

Results

Conclusion