A UAF1-Containing Multisubunit Protein Complex Regulates the Fanconi Anemia Pathway

Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA.
Molecular Cell (Impact Factor: 14.02). 01/2008; 28(5):786-97. DOI: 10.1016/j.molcel.2007.09.031
Source: PubMed


The deubiquitinating enzyme USP1 controls the cellular levels of the DNA damage response protein Ub-FANCD2, a key protein of the Fanconi anemia DNA repair pathway. Here we report the purification of a USP1 multisubunit protein complex from HeLa cells containing stoichiometric amounts of a WD40 repeat-containing protein, USP1 associated factor 1 (UAF1). In vitro reconstitution of USP1 deubiquitinating enzyme activity, using either ubiquitin-7-amido-4-methylcoumarin (Ub-AMC) or purified monoubiquitinated FANCD2 protein as substrates, demonstrates that UAF1 functions as an activator of USP1. UAF1 binding increases the catalytic turnover (kcat) but does not increase the affinity of the USP1 enzyme for the substrate (KM). Moreover, we show that DNA damage results in an immediate shutoff of transcription of the USP1 gene, leading to a rapid decline in the USP1/UAF1 protein complex. Taken together, our results describe a mechanism of regulation of the deubiquitinating enzyme, USP1, and of DNA repair.

Full-text preview

Available from:
  • Source
    • "The deubiquitinating enzyme USP1 regulates the level of FANCD2-Ub (Nijman et al., 2005). USP1 associates with its activating factor UAF1, and the USP1-UAF1 complex removes monoubiquitin from FANCD2 to complete the repair (Cohn et al., 2007) (Fig. 1D). In addition to its stimulatory role, UAF1 is also necessary for recruiting the FANCD2-FANCI complex to USP1 (Yang et al., 2011). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Genome instability, primarily caused by faulty DNA repair mechanisms, drives tumorigenesis. Therapeutic interventions that exploit deregulated DNA repair in cancer have made considerable progress by targeting tumor-specific alterations of DNA repair factors, which either induces synthetic lethality or augments the efficacy of conventional chemotherapy and radiotherapy. The study of Fanconi anemia (FA), a rare inherited blood disorder and cancer predisposition syndrome, has been instrumental in understanding the extent to which DNA repair defects contribute to tumorigenesis. The FA pathway functions to resolve blocked replication forks in response to DNA interstrand cross-links (ICLs), and accumulating knowledge of its activation by the ubiquitin-mediated signaling pathway has provided promising therapeutic opportunities for cancer treatment. Here, we discuss recent advances in our understanding of FA pathway regulation and its potential application for designing tailored therapeutics that take advantage of deregulated DNA ICL repair in cancer.
    Moleculer Cells 07/2015; 38(8). DOI:10.14348/molcells.2015.0175 · 2.09 Impact Factor
  • Source
    • "The DUB activity of USP46 and USP12 has been proven in vitro (Quesada et al. 2004; Cohn et al. 2009; Kee et al. 2010). In C. elegans, USP46 and USP12 both bind to USP1-associated factor 1 (UAF1/WDR48) and the binding dramatically enhances the activity of USP12 and USP46 (Cohn et al. 2007, 2009; Dahlberg and Juo 2014). Our findings indicate that USP46 function is well conserved among species. "

    Journal of Neurochemistry 01/2015; · 4.28 Impact Factor
  • Source
    • "The observation that autocleaved USP1 remains active was unexpected, since the cleavage event disrupts its His box catalytic motif. In this regard, it has been proposed that USP1NT and USP1CT fragments may be held together by UAF1 forming a catalytically competent ternary complex [58]. This model, however, seems difficult to reconcile with the finding that the carboxy-terminal region of USP1 is unable to interact with UAF1 [73], and would require further experimental validation. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Reversible protein ubiquitination is emerging as a key process for maintaining cell homeostasis, and the enzymes that participate in this process, in particular E3 ubiquitin ligases and deubiquitinases (DUBs), are increasingly being regarded as candidates for drug discovery. Human DUBs are a group of approximately 100 proteins, whose cellular functions and regulatory mechanisms remain, with some exceptions, poorly characterized. One of the best-characterized human DUBs is ubiquitin-specific protease 1 (USP1), which plays an important role in the cellular response to DNA damage. USP1 levels, localization and activity are modulated through several mechanisms, including protein-protein interactions, autocleavage/degradation and phosphorylation, ensuring that USP1 function is carried out in a properly regulated spatio-temporal manner. Importantly, USP1 expression is deregulated in certain types of human cancer, suggesting that USP1 could represent a valid target in cancer therapy. This view has gained recent support with the finding that USP1 inhibition may contribute to revert cisplatin resistance in an in vitro model of non-small cell lung cancer (NSCLC). Here, we describe the current knowledge on the cellular functions and regulatory mechanisms of USP1. We also summarize USP1 alterations found in cancer, combining data from the literature and public databases with our own data. Finally, we discuss the emerging potential of USP1 as a target, integrating published data with our novel findings on the effects of the USP1 inhibitor pimozide in combination with cisplatin in NSCLC cells.
    Molecular Cancer 08/2013; 12(1):91. DOI:10.1186/1476-4598-12-91 · 4.26 Impact Factor
Show more