Abstract PR4: Regulation of the Fanconi anemia pathway by a SUMO-like delivery network

Kailin Yang; George-Lucian Moldovan; Patrizia Vinciguerra; Junko Murai; Shunichi Takeda; Alan D. D'Andrea

doi:10.1158/1538-7445.FBCR11-PR4

Abstract Objective: The USP1/UAF1 complex is a critical regulator of the FA pathway. The USP1/UAF1 complex deubiquitinates the Fanconi Anemia proteins, FANCD2/FANCI, and promotes homologous recombination and DNA crosslink repair. USP1/UAF1 also deubiquitinates PCNA-Ub, a key player in translesion DNA synthesis. This step requires the PCNA interacting protein, hELG1. The goal of our study is to identify the molecular mechanism of USP1/UAF1 complex targeting to the FANCD2-Ub/FANCI-Ub heterodimer and tothePCNA-Ub/hELG1 complex. Methods: Using bioinformatics, we identified a tandem repeat of SUMO-like domains (SLD) in the C-terminus of UAF1 (SLD1 and SLD2). A SLD sequence is a domain of approximately 100 amino acids with significant sequence identity and homology to the small ubiquitin-like modifier (SUMO). We tested the function of a mutant form of UAF1 lacking the SLD2 domain. Interestingly, this mutant UAF1 protein bound and stimulated the deubiquitinating activity of USP1 but failed to complement UAF1 knockout DT40 cells. We then discovered a SUMO-like-domain interacting motif (SIM) on both FANCI and hELG1. We therefore investigated the functional importance of the SLD-SIM interaction in regulating the Fanconi anemia pathway. Results: The SLD2 domain of UAF1 is dispensable for activating USP1 in vitro. However, complementation analyses using UAF1 knockout DT40 cells showed that the SLD2 is critical for the deubiquitination of USP1 substrates in vivo and for homologous recombination. We demonstrated that the SLD2 domain of UAF1 binds directly to the SIM sequence on FANCI and hELG1, and thereby regulates the deubiquitination of FANCD2-Ub and PCNA-Ub respectively. Disruption of these SLD-SIM interactions blocked the deubiquitination of FANCD2-Ub and/or PCNA-Ub, rendering cells DNA repair deficient. We also modeled the 3-D structure of SLD2 and identified critical residues involved in SLD-SIM interaction. Conclusions: Our study demonstrates that unique SLD-SIM interactions mediate the targeting of the USP1/UAF1 deubiquitinating complex to its monoubiquitinated substrates, FANCD2/FANCI and hELG1/PCNA, and thereby coordinate homologous recombination. This is a novel mechanism of DNA repair regulation, and it may have relevance to other DNA repair pathways. The SLD-SIM interaction sites are potential drug target sites for agonists or antagonists of the FA pathway. They may be useful in screening drugs for the treatment of FA patients and cancer patients from the general (non-FA) population. This abstract is also presented as Poster B38. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr PR4.

Abstract PR4: Regulation of the Fanconi anemia pathway by a SUMO-like delivery network

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