Studying rare human genetic diseases often leads to a better understanding of normal cellular functions. Fanconi anemia (FA), for example, has elucidated a novel DNA repair mechanism required for maintaining genomic stability and preventing cancer. The FA pathway, an essential tumor-suppressive pathway, is required for protecting the human genome from a specific type of DNA damage; namely, DNA interstrand cross-links (ICLs). In this review, we discuss the recent progress in the study of the FA pathway, such as the identification of new FANCM-binding partners and the identification of RAD51C and FAN1 (Fanconi-associated nuclease 1) as new FA pathway-related proteins. We also focus on the role of the FA pathway as a potential regulator of DNA repair choices in response to double-strand breaks, and its novel functions during the mitotic phase of the cell cycle.

A balance between ubiquitination and deubiquitination regulates numerous cellular processes and pathways, and specific deubiquitinating enzymes often play the decisive role of controlling this balance. We recently reported that the USP1 deubiquitinating enzyme, which regulates the Fanconi anemia pathway by deubiquitinating the central player of the pathway, FANCD2, is activated by the WD40-repeat containing UAF1 protein through formation of a stable USP1/UAF1 protein complex. Here we present the isolation of two novel multisubunit deubiquitinating enzyme complexes containing USP12 and USP46, respectively. Both complexes contain the UAF1 protein as a bona fide subunit. Interestingly, UAF1 regulates the enzymatic activity of both enzyme complexes, suggesting that this activator protein may regulate a subclass of human deubiquitinating enzymes. We postulate that additional WD40-containing proteins may also form complexes with other human deubiquitinating enzymes and thereby regulate their activity and substrate specificity.Ubiquitination and deubiquitination regulate a number of essential biological processes such as gene transcription, DNA replication, and DNA repair (1). Ubiquitin modifications can be divided into three principally different types. First, monoubiquitination may alter the activity of the substrate, as described for the FANCD2 protein of the Fanconi anemia (FA) 2 pathway (2) and the PCNA protein involved in Trans Lesion Synthesis (2, 3), or may alter the cellular localization of the protein (4). Second, polyubiquitination through K48-linkage typically targets the protein substrate for degradation by the proteasome (5). Third, polyubiquitination through the K63-linkage can alter the activity of the protein by modifying its protein-protein interaction properties. A recent example of K63 polyubiquitination is that of the histone variant H2AX, which is polyubiquitinated in response to DNA damage, and as such, is believed to orchestrate the recruitment of DNA repair factors to sites of DNA damage on the chromatin (6).Processes regulated by ubiquitination are often controlled by the opposing enzymatic reaction, namely deubiquitination. First, accurate deubiquitination of the FANCD2 protein by the USP1/UAF1 complex is essential for an intact Fanconi anemia pathway. Loss of USP1 activity leads to accumulation of monoubiquitinated FANCD2, dysregulation of the FA pathway, and cellular hypersensitivity to DNA cross-linking agents (7-9). Second, failure to deubiquitinate Cdc20 as part of the APC-inhibitory Mad2-Cdc20 complex by USP44, leads to an anaphase entry defect (10). Third, the USP22 deubiquitinating enzyme, as a subunit of the SAGA complex, is critical for appropriate progression through the cell cycle due to its function in transcriptional regulation by deubiquitinating monoubiquitinated histone H2B (11).There are ϳ95 deubiquitinating enzymes in human (12). The family of deubiquitinating enzymes is divided into five subfamilies, including the USP subfamily (58 members), the UCH subfamily (4 members), ...

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Saccharomyces cerevisiae Rsp5 is an essential HECT ubiquitin ligase involved in several biological processes. To gain further insight into regulation of this enzyme, we identified proteins that copurified with epitope-tagged Rsp5. Ubp2, a deubiquitinating enzyme, was a prominent copurifying protein. Rup1, a previously uncharacterized UBA domain protein, was required for binding of Rsp5 to Ubp2 both in vitro and in vivo. Overexpression of Ubp2 or Rup1 in the rsp5-1 mutant elicited a strong growth defect, while overexpression of a catalytically inactive Ubp2 mutant or Rup1 deleted of the UBA domain did not, suggesting an antagonistic relationship between Rsp5 and the Ubp2/Rup1 complex. Consistent with this model, rsp5-1 temperature sensitivity was suppressed by either ubp2D or rup1D mutations. Ubp2 reversed Rsp5-catalyzed substrate ubiquitination in vitro, and Rsp5 and Ubp2 preferentially assembled and disassembled, respectively, K63-linked polyubiquitin chains. Together, these results indicate that Rsp5 activity is modulated by being physically coupled to the Rup1/Ubp2 deubiquitinating enzyme complex, representing a novel mode of regulation for an HECT ubiquitin ligase.

IntroductionThe 13 identified Fanconi anemia (FA) proteins cooperate in a common cellular pathway regulating the cellular response to DNA cross-linking agents, such as cisplatin (CDDP), diepoxybutane (DEB), and mitomycin C (MMC). 1 Of these FA proteins, 8 (A, B, C, E, F, G, L, and M) are assembled into a core complex, 2,3 which contains a ubiquitin E3 ligase activity (FANCL subunit) 4 and a DNA translocase activity (FANCM). 5 In response to DNA damage, or during S-phase progression, the FA core complex coordinately monoubiquitinates 2 downstream substrates, FANCD2 6,7 and FANCI. [8][9][10] These monoubiquitinated proteins subsequently translocate to the chromatin where they are believed to interact with additional downstream FA proteins, including FANCJ/BRIP1, [11][12][13] FANCD1/BRCA2, 14 and FANCN/PALB2. 15,16 These downstream proteins then regulate homologous recombination (HR) repair. Disruption of any of the proteins in the FA pathway accounts for the common cellular and clinical phenotype of FA patients. 17 How the pathway participates in the process of DNA cross-link repair remains unknown. 18 Some FA complementation groups also exhibit additional phenotypic variation, suggesting that some FA genes have functions outside a simple linear FA pathway. [19][20][21][22] The FA core complex may have additional functions beyond the monoubiquitination of FANCD2 and FANCI. 8 A FANCD2-Ub linear fusion protein complements the MMC hypersensitivity of Fancd2-deficient chicken cells, but fails to correct the phenotype of FA core complex-deficient cells. 23 The FA core complex may therefore have additional activities, such as the recognition of specific DNA substrates, the regulation of the DNA replication machinery, and/or the monoubiquitination of additional (unknown) substrates. These additional functions may be explained, at least in part, by the presence of FA core subcomplexes with variable sizes and variable subcellular distributions. 3 When a replication fork encounters an interstrand DNA crosslink during replication, the replication fork arrests near the lesion, resulting in aberrant DNA structures. These abnormal structures activate checkpoint and repair pathways. FA cells, carrying mutations in FA genes, are highly sensitive to DNA crosslinking agents, compared with other DNA-damaging agents, such as ionizing radiation (IR), ultraviolet (UV), and hydroxyurea (HU). This hypersensitivity suggests that some components of the FA core complex may be involved in detecting and binding the DNA lesions caused by treatment of DNA crosslinking agents.The recently identified FANCM 5 and FANCJ 11-13 proteins suggest a direct involvement of FA proteins at sites of DNA repair. FANCM is homologous to the archaeal protein Hef (helicaseassociated endonuclease for fork-structured DNA), and is a member of the XP-F superfamily. 24 The N-terminal region of FANCM is able to bind to single-stranded DNA. 25 Moreover, FANCM has a DNA-dependent ATPase activity, and it can dissociate DNA triple helices in vitro. 5,25 FANCJ/BRIP1, whi...