Deubiquitination of FANCD2 Is Required for DNA Crosslink Repair

Abstract

SummaryMonoubiquitination of FANCD2 and PCNA promotes DNA repair. It causes chromatin accumulation of FANCD2 and facilitates PCNA's recruitment of translesion polymerases to stalled replication. USP1, a protease that removes monoubiquitin from FANCD2 and PCNA, was thought to reverse the DNA damage response of these substrates. We disrupted USP1 in chicken cells to dissect its role in a stable genetic system. USP1 ablation increases FANCD2 and PCNA monoubiquitination but unexpectedly results in DNA crosslinker sensitivity. This defective DNA repair is associated with constitutively chromatin-bound, monoubiquitinated FANCD2. In contrast, persistent PCNA monoubiquitination has negligible impact on DNA repair or mutagenesis. USP1 was previously shown to autocleave after DNA damage. In DT40, USP1 autocleavage is not stimulated by DNA damage, and expressing a noncleavable mutant in the USP1 knockout strain partially rescues crosslinker sensitivity. We conclude that efficient DNA crosslink repair requires FANCD2 deubiquitination, whereas FANCD2 monoubiquitination is not dependent on USP1 autocleavage.

The Rap80-BRCC36 de-ubiquitinating enzyme complex antagonizes RNF8-Ubc13-dependent ubiquitination events at DNA double strand breaks

“…De-ubiquitination of the Fanconi Anemia D2 protein, FANCD2, by the USP1 DUB represents a second example in which DUB activity is necessary to terminate DNA damage-inducible ubiquitination events (30). Failure to either ubiquitinate FANCD2 or to de-ubiquitinate FANCD2-Ub confers sensitivity to DNA cross-linking agents (31,32).…”

“…The PP2A phosphatase dephosphorylates ␥H2AX, and indeed, PP2A deficiency renders cells IR-sensitive (33). Additionally, sustained levels of mono-ubiquitinated FANCD2 in USP1-null cells confers reduced viability after DNA damage (31). Thus, opposing activation and termination responses on ␥H2AX and other proteins involved in DNA repair appear to be crucial to maintain viability after DNA damage.…”

Regulation of DNA cross-link repair by the Fanconi anemia/BRCA pathway

“…1G; Nijman et al 2005;Cohn et al 2007). Of note, genetic disruption of murine Usp1 results in a phenotype similar to FA, and both Usp1 and Uaf1 knockout chicken DT40 cells exhibit hypersensitivity to DNA cross-linking agents (Oestergaard et al 2007;Kim et al 2009;Murai et al 2011). Therefore, USP1-dependent deubiquitination constitutes another critical repair step in the completion of DNA ICL repair.…”

A novel role for the deubiquitinase USP1 in the control of centrosome duplication

“…11 USP1 has been identified as a key regulator in the DNA repair processes, mainly in the Fanconi anemia pathway and Translesion DNA synthesis by regulating ubiquitination status of FANCD2 and PCNA, 12,13 Mono-ubiquitinated FANCD2 and PCNA serve as a platform to recruit DNA repair proteins to DNA damage sites, [14][15][16] and USP-induced deubiquitination of FANCD2 and PCNA is crucial for the correct function of DNA repair pathway. 17,18 Indeed, Usp1 gene deletion in mice 18 and USP1 inhibition by small molecules displayed chemosensitizing effects against DNA damaging agents, [19][20][21] indicating that USP1 is required for an efficient DNA repair activity. Besides DNA repair-related function, recent study reveals that increased level of USP1 is found in human osteosarcoma cell lines.…”