MRE11–RAD50–NBS1 is a critical regulator of FANCD2 stability and function during DNA double-strand break repair

Abstract

Monoubiquitination of the Fanconi anaemia protein FANCD2 is a key event leading to repair of interstrand cross-links. It was reported earlier that FANCD2 co-localizes with NBS1. However, the functional connection between FANCD2 and MRE11 is poorly understood. In this study, we show that inhibition of MRE11, NBS1 or RAD50 leads to a destabilization of FANCD2. FANCD2 accumulated from mid-S to G2 phase within sites containing single-stranded DNA (ssDNA) intermediates, or at sites of DNA damage, such as those created by restriction endonucleases and laser irradiation. Purified FANCD2, a ring-like particle by electron microscopy, preferentially bound ssDNA over various DNA substrates. Inhibition of MRE11 nuclease activity by Mirin decreased the number of FANCD2 foci formed in vivo. We propose that FANCD2 binds to ssDNA arising from MRE11-processed DNA double-strand breaks. Our data establish MRN as a crucial regulator of FANCD2 stability and function in the DNA damage response.

Preventing over-resection by DNA2 helicase/nuclease suppresses repair defects in Fanconi anemia cells

“…45,50 CtIP or Mre11 appear to be involved in an early step in ICL repair, because CtIP or Mre11 depletion prevents FANCD2 recruitment to sites of damage and ubiquitylation of FANCD2 in the presence of ICL damage. 23,42 Since CtIP and Mre11 are implicated in resection just upstream of DNA2, 29,31,32,51 it was of interest to test whether DNA2 was also required at an early step for FANCD2 activation. Previously, we showed that the depletion of endogenous DNA2 did not affect FANCD2 monoubiquitination after cisplatin treatment.…”

“…23 Thus, CtIP and MRN complex play a role early in the FA/BRCA pathway in addition to their critical role in the HDR step required for the completion of FA/BRCA repair. In summary, the interaction of FA/BRCA factors with so many additional DNA repair factors and pathways suggests that the FA pathway may be the central sensor and/or regulator for most cellular DNA damage responses.…”

ATM protein kinase: the linchpin of cellular defenses to stress

“…It seems that components of the MRN complex can also influence the stability of other proteins involved in DNA damage response. For example, MRN is an indispensable for stabilization of FANCD2 during DNA repair as silencing of MRN by the specific inhibitor Mirin influenced accumulation of FANCD2 at the sites of DNA DSBs [118].…”

Crosstalk between translesion synthesis, Fanconi anemia network, and homologous recombination repair pathways in interstrand DNA crosslink repair and development of chemoresistance

“…This suggests that BRCA1 and CtIP play a role downstream of the FA core complex allowing for FANCD2 foci formation at the site of damage. A role for the MRN complex in FANCD2 stability has also been reported as silencing of MRE11, RAD50 or NBS1 leads to decrease in FANCD2 protein life [74]. Together these data suggest that integral members of HRR are needed for proper FANCD2 function within the FA network.…”