Faculty Bibliography

Deubiquitinating enzymes (DUBs) recognize and cleave linkage-specific polyubiquitin (polyUb) chains, but mechanisms underlying specificity remain elusive in many cases. The severe acute respiratory syndrome (SARS) coronavirus papain-like protease (PLpro) is a DUB that cleaves ISG15, a two-domain Ub-like protein, and Lys48-linked polyUb chains, releasing diUb(Lys48) products. To elucidate this specificity, we report the 2.85 A crystal structure of SARS PLpro bound to a diUb(Lys48) activity-based probe. SARS PLpro binds diUb(Lys48) in an extended conformation via two contact sites, S1 and S2, which are proximal and distal to the active site, respectively. We show that specificity for polyUb(Lys48) chains is predicated on contacts in the S2 site and enhanced by an S1-S1' preference for a Lys48 linkage across the active site. In contrast, ISG15 specificity is dominated by contacts in the S1 site. Determinants revealed for polyUb(Lys48) specificity should prove useful in understanding PLpro deubiquitinating activities in coronavirus infections.

Both proteolytic and nonproteolytic functions of ubiquitination are essential regulatory mechanisms for promoting DNA repair and the DNA damage response in mammalian cells. Deubiquitinating enzymes (DUBs) have emerged as key players in the maintenance of genome stability. In this minireview, we discuss the recent findings on human DUBs that participate in genome maintenance, with a focus on the role of DUBs in the modulation of DNA repair and DNA damage signaling.

Fanconi anemia (FA) is a rare human genetic disorder characterized by developmental defects, bone marrow failure and cancer predisposition, primarily due to a deficiency in the repair of DNA interstrand crosslinks (ICLs). ICL repair through the FA DNA repair pathway is a complicated multi-step process, involving at least 19 FANC proteins and coordination of multiple DNA repair activities, including homologous recombination, nucleotide excision repair and translesion synthesis (TLS). SUMOylation is a critical regulator of several DNA repair pathways, however, the role of this modification in controlling the FA pathway is poorly understood. Here, we summarize recent advances in the fine-tuning of the FA pathway by small ubiquitin-like modifier (SUMO)-targeted ubiquitin ligases (STUbLs) and other SUMO-related interactions, and discuss the implications of these findings in the design of novel therapeutics for alleviating FA-associated condition, including cancer.

Excess dormant origins bound by the minichromosome maintenance (MCM) replicative helicase complex play a critical role in preventing replication stress, chromosome instability, and tumorigenesis. In response to DNA damage, replicating cells must coordinate DNA repair and dormant origin firing to ensure complete and timely replication of the genome; how cells regulate this process remains elusive. Herein, we identify a member of the Fanconi anemia (FA) DNA repair pathway, FANCI, as a key effector of dormant origin firing in response to replication stress. Cells lacking FANCI have reduced number of origins, increased inter-origin distances, and slowed proliferation rates. Intriguingly, ATR-mediated FANCI phosphorylation inhibits dormant origin firing while promoting replication fork restart/DNA repair. Using super-resolution microscopy, we show that FANCI co-localizes with MCM-bound chromatin in response to replication stress. These data reveal a unique role for FANCI as a modulator of dormant origin firing and link timely genome replication to DNA repair.

DNA damage associated with viral DNA synthesis can result in double-strand breaks that threaten genome integrity and must be repaired. Here, we establish that the cellular Fanconi anemia (FA) genomic stability pathway is exploited by herpes simplex virus 1 (HSV-1) to promote viral DNA synthesis and enable its productive growth. Potent FA pathway activation in HSV-1-infected cells resulted in monoubiquitination of FA effector proteins FANCI and FANCD2 (FANCI-D2) and required the viral DNA polymerase. FANCD2 relocalized to viral replication compartments, and FANCI-D2 interacted with a multisubunit complex containing the virus-encoded single-stranded DNA-binding protein ICP8. Significantly, whereas HSV-1 productive growth was impaired in monoubiquitination-defective FA cells, this restriction was partially surmounted by antagonizing the DNA-dependent protein kinase (DNA-PK), a critical enzyme required for nonhomologous end-joining (NHEJ). This identifies the FA-pathway as a cellular factor required for herpesvirus productive growth and suggests that FA-mediated suppression of NHEJ is a fundamental step in the viral life cycle.

The ubiquitin-modification status of proteins in cells is highly dynamic and maintained by specific ligation machineries (E3 ligases) that tag proteins with ubiquitin or by deubiquitinating enzymes (DUBs) that remove the ubiquitin tag. The development of tools that offset this balance is critical in characterizing signaling pathways that utilize such ubiquitination switches. Herein, we generated a DUB-resistant ubiquitin mutant that is recalcitrant to cleavage by various families of DUBs both in vitro and in mammalian cells. As a proof-of-principle experiment, ectopic expression of the uncleavable ubiquitin stabilized monoubiquitinated PCNA in the absence of DNA damage and also revealed a defect in the clearance of the DNA damage response at unprotected telomeres. Importantly, a proteomic survey using the uncleavable ubiquitin identified ubiquitinated substrates, validating the DUB-resistant ubiquitin expression system as a valuable tool for interrogating cell signaling pathways.

Deamidation of N-terminal Gln by the Ntaq1 Nt(Q)-amidase is a part of the Arg/N-end rule pathway, a ubiquitin-dependent proteolytic system. Here we identify Gln-Usp1(Ct), the C-terminal fragment of the autocleaved Usp1 deubiquitylase, as the first physiological Arg/N-end rule substrate that is targeted for degradation through deamidation of N-terminal Gln. Usp1 regulates genomic stability, in part through the deubiquitylation of monoubiquitylated PCNA, a DNA polymerase processivity factor. The autocleaved Usp1 remains a deubiquitylase because its fragments remain associated with Uaf1, an enhancer of Usp1 activity, until the Gln-Usp1(Ct) fragment is selectively destroyed by the Arg/N-end rule pathway. We also show that metabolic stabilization of Gln-Usp1(Ct) results in a decreased monoubiquitylation of PCNA and in a hypersensitivity of cells to ultraviolet irradiation. Thus, in addition to its other functions in DNA repair and chromosome segregation, the Arg/N-end rule pathway regulates genomic stability through the degradation-mediated control of the autocleaved Usp1 deubiquitylase.

Deubiquitinating enzymes (DUBs) constitute a large family of cysteine proteases that have a broad impact on numerous biological and pathological processes, including the regulation of genomic stability. DUBs are often assembled onto multiprotein complexes to assist in their localization and substrate selection, yet it remains unclear how the enzymatic activity of DUBs is modulated by intracellular signals. Herein, we show that bursts of reactive oxygen species (ROS) reversibly inactivate DUBs through the oxidation of the catalytic cysteine residue. Importantly, USP1, a key regulator of genomic stability, is reversibly inactivated upon oxidative stress. This, in part, explains the rapid nature of PCNA monoubiquitination-dependent DNA damage tolerance in response to oxidative DNA damage in replicating cells. We propose that DUBs of the cysteine protease family act as ROS sensors in human cells and that ROS-mediated DUB inactivation is a critical mechanism for fine-tuning stress-activated signaling pathways.

Interstand crosslinks (ICLs) are DNA lesions where the bases of opposing DNA strands are covalently linked, inhibiting critical cellular processes such as transcription and replication. Chemical agents that generate ICLs cause chromosomal abnormalities including breaks, deletions and rearrangements, making them highly genotoxic compounds. This toxicity has proven useful for chemotherapeutic treatment against a wide variety of cancer types. The majority of our understanding of ICL repair in humans has been uncovered through analysis of the rare genetic disorder Fanconi anemia, in which patients are extremely sensitive to crosslinking agents. Here, we discuss recent insights into ICL repair gained using new repair assays and highlight the role of the Fanconi anemia repair pathway during replication stress.