Phosphorylation of FANCD2 Inhibits the FANCD2/FANCI Complex and Suppresses the Fanconi Anemia Pathway in the Absence of DNA Damage

David Lopez-Martinez,Marian Kupculak,Di Yang,Yasunaga Yoshikawa,Chih-Chao Liang,Ronghu Wu,Steven P Gygi,Martin A Cohn

doi:10.1016/j.celrep.2019.05.003

David Lopez-Martinez, Marian Kupculak + Show 6 more

Open Access

https://doi.org/10.1016/j.celrep.2019.05.003

Copy DOI

Journal: Cell Reports	Publication Date: Jun 1, 2019
Citations: 29	License type: cc-by

Affiliation: Harvard University, University of Oxford

Abstract

SummaryInterstrand crosslinks (ICLs) of the DNA helix are a deleterious form of DNA damage. ICLs can be repaired by the Fanconi anemia pathway. At the center of the pathway is the FANCD2/FANCI complex, recruitment of which to DNA is a critical step for repair. After recruitment, monoubiquitination of both FANCD2 and FANCI leads to their retention on chromatin, ensuring subsequent repair. However, regulation of recruitment is poorly understood. Here, we report a cluster of phosphosites on FANCD2 whose phosphorylation by CK2 inhibits both FANCD2 recruitment to ICLs and its monoubiquitination in vitro and in vivo. We have found that phosphorylated FANCD2 possesses reduced DNA binding activity, explaining the previous observations. Thus, we describe a regulatory mechanism operating as a molecular switch, where in the absence of DNA damage, the FANCD2/FANCI complex is prevented from loading onto DNA, effectively suppressing the FA pathway.

Highlights

Our genomes are exposed to multiple sources of DNA damage, both exogenous and endogenous
FANCD2 Is Phosphorylated on Residues S882, T884, S886, S891, T896, and S898 In Vivo In order to study phosphorylation events in FANCD2, we introduced a Flag-HA tag into the FANCD2 gene in HeLa cells using CRISPR/Cas9, creating an N-terminal-tagged fusion protein (Figures 1A and S1A)
Examination of the crystal structure of the mouse FANCD2/FANCI complex (Joo et al, 2011) and the cryoelectron microscopy structure of the human FANCD2/FANCI complex (Liang et al, 2016) suggests that the 882–898 phosphorylation cluster is located on the surface of the inner cavity of the heterodimer, potentially in contact with DNA (Figure 1F)

Summary

Introduction

Our genomes are exposed to multiple sources of DNA damage, both exogenous and endogenous. The cell counts on multiple repair pathways to handle these damages, which are strictly regulated by a complex signaling network involving different posttranslational modifications called the DNA damage response. A complex set of DNA damage response pathways handles ICLs, including the Fanconi anemia (FA)/BRCA (breast cancer genes) repair pathway, translesion synthesis (TLS), homologous recombination (HR), and nucleotide excision repair (NER) (Ceccaldi et al, 2016; Lopez-Martinez et al, 2016) and a separate NEIL3-dependent ICL repair pathway (Semlow et al, 2016). 22 genes have been identified to cause FA, and they all code for proteins implicated in the repair of ICLs. Mutations in several of these genes have been linked to increased incidence of certain cancers, for example, BRCA1, BRCA2 and PALB2 (Deans and West, 2011; King et al, 2003)

Methods

Results

Discussion

Conclusion