A DUB less step? Tighten up D loop
DNA double strand breaks (DSBs) can arise through exposure to exogenous DNA damaging agents such as ionizing radiation, as well as through endogenous means; for example, via DNA replication fork collapse. Irrespective of the source, the physical severing of the sugar phosphate backbone represents an acute threat to organismal viability and genome stability. Hanahan and Weinberg describe genome instability and mutation as an enabling characteristic of cancer. Indeed, chromosome structural rearrangements, which are pervasive in cancer, invariably arise from DSB repair gone awry. To deal with this threat, all cells have evolved 2 principal means to repair DSBs: homologous recombination (HR) and nonhomologous DNA end joining (NHEJ) HR is predominantly a conservative and error free process, employing a homologous template, usually the sister chromatid, to repair the break. RAD51, the eukaryotic ortholog of bacterial RecA, is the major HR protein. Conversely, NHEJ is typically error prone, and rejoins the break without regard for the state of the ends, often resulting in loss of nucleotides or the re joining of noncontiguous ends. Exemplifying the importance of HR, many key tumor suppressor genes encode central HR players, e.g. BRCA1 and BRCA2. Furthermore, several genetic diseases characterized by increased cancer risk are caused by mutations in HR genes, one example of which is Fanconi anemia (FA) FA is clinically characterized by congenital defects, bone marrow failure, and increased cancer risk. FA is caused by mutation of any one of 20 known genes, which encode proteins that function cooperatively in the FA BRCA pathway to promote HR. The molecular links between FA and HR are an area of active investigation. Evidence presented in this volume of Cell Cycle points to a novel noncanonical connection between enzymes involved in the major regulatory step of the FA BRCA pathway and a key HR effector. This regulatory step is the site specific monoubiquitination of the FANCD2 and FANCI proteins. The E2 ubiquitin conjugating enzyme FANCT/UBE2T and the E3 ubiquitin ligase FANCL catalyze the forward step of this reaction. The reverse step deubiquitination is catalyzed by the USP1 deubiquitinating enzyme (DUB) and its heterodimeric binding partner UAF1. In a large scale global proteomic screen of DUB interacting networks, Sowa et al. previously determined that the USP1UAF1 heterodimer interacts with the RAD51AP1 protein. RAD51AP1 is a vertebrate specific accessory factor for RAD51 that promotes the assembly of the synaptic complex and D (displacement) loop, key HR intermediate structures (Fig. 1) However, the functional significance of this interaction was not examined. In the accompanying Cell Cycle manuscript, Cukras et al. have tackled this important question. The authors verified this interaction and, by depleting UAF1 using siRNA, established that the interaction between USP1 and RAD51AP1 is UAF1 dependent. The authors also established that the UAF1 WD40 repeats as well as its SUMO like domains (SLDs) are necessary for RAD51AP1 binding. Previous studies had demonstrated that USP1 regulates the stability of the ID (inhibitor of DNA binding) proteins. Similarly, Cukras et al. show that depletion of USP1 or UAF1 leads to destabilization of RAD51AP1. Cukras et al. next sought to map the region of RAD51AP1 that binds to UAF1. Serial truncations and mutagenesis analysis established that residues D133 L137 are required for efficient RAD51AP1 UAF1 binding. Accordingly, deletion of this UAF1 binding region (DDYLDL) resulted in decreased RAD51AP1 stability, supporting the theory that USP1 UAF1 RAD51AP1 form a stable protein complex. Interestingly, mutation of RAD51AP1 K139, previously shown to be a site of ubiquitination, did not affect interaction with UAF1. To explore the functional significance of the RAD51AP1 UAF1 interaction, Cukras et al. expressed wild type or RAD51AP1 DDYLDL in U2OS cells depleted of endogenous RAD51AP1. In contrast to wild type RAD51AP1, the DDYLDLmutant failed to correct cellular ICL sensitivity. Furthermore, RAD51AP1 DDYLDL expressing cells exhibited persistent DNA damage inducible RAD51 nuclear foci, suggesting that the USP1 UAF1 RAD51AP1 complex may promote the efficient and timely resolution of a key HR intermediate structure. A recent complementary study in Cell Reports by Liang et al. provides further insight into the functional significance of the RAD51AP1 UAF1 interaction. Similar to Cukras et al. Liang et al. establish that the UAF1 SLDs mediate interaction with RAD51AP1. While mutation of these SLDs compromises interaction with RAD51AP1, these mutants are proficient for
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