Faculty Bibliography

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.

Tight regulation of the cell cycle and DNA repair machinery is essential for maintaining genome stability. The APC/CCdh1 ubiquitin ligase complex is a key regulator of protein stability during the G 1 phase of the cell cycle. APC/CCdh1 regulates and promotes the degradation of proteins involved in both cell cycle regulation and DNA repair. In a recent study, we identified a novel APC/CCdh1 substrate, the ubiquitin protease USP1. USP1 is a critical regulator of both the Fanconi anemia (FA) and translesion synthesis (TLS) DNA repair pathways. Here, we provide additional mechanistic insights into the regulation of USP1 during the cell cycle. Specifically, we demonstrate that USP1 is phosphorylated in mitosis by cyclin-dependent kinases (Cdks), and that this phosphorylation event may prevent premature degradation of USP1 during normal cell cycle progression. Finally, we provide a unifying hypothesis integrating the role of G 1-specific proteolysis of USP1 with the regulation of the transcriptional repressors, Inhibitor of DNA-binding (ID) proteins

Targeted protein destruction of critical cellular regulators during the G1 phase of the cell cycle is achieved by anaphase-promoting complex/cyclosome(Cdh1) (APC/C(Cdh1)), a multisubunit E3 ubiquitin ligase. Cells lacking Cdh1 have been shown to accumulate deoxyribonucleic acid (DNA) damage, suggesting that it may play a previously unrecognized role in maintaining genomic stability. The ubiquitin-specific protease 1 (USP1) is a known critical regulator of DNA repair and genomic stability. In this paper, we report that USP1 was degraded in G1 via APC/C(Cdh1). USP1 levels were kept low in G1 to provide a permissive condition for inducing proliferating cell nuclear antigen (PCNA) monoubiquitination in response to ultraviolet (UV) damage before DNA replication. Importantly, expression of a USP1 mutant that cannot be degraded via APC/C(Cdh1) inhibited PCNA monoubiquitination during G1, likely compromising the recruitment of trans-lesion synthesis polymerase to UV repair sites. Thus, we propose a role for APC/C(Cdh1) in modulating the status of PCNA monoubiquitination and UV DNA repair before S phase entry

The N-terminal nuclear export sequence (NES) of inhibitor of nuclear factor kappa B (NF-kappaB) alpha (IkappaBalpha) promotes NF-kappaB export from the cell nucleus to the cytoplasm, but the physiological role of this export regulation remains unknown. Here we report the derivation and analysis of genetically targeted mice harboring a germline mutation in IkappaBalpha NES. Mature B cells in the mutant mice displayed nuclear accumulation of inactive IkappaBalpha complexes containing a NF-kappaB family member, cRel, causing their spatial separation from the cytoplasmic IkappaB kinase. This resulted in severe reductions in constitutive and canonical NF-kappaB activities, synthesis of p100 and RelB NF-kappaB members, noncanonical NF-kappaB activity, NF-kappaB target gene induction, and proliferation and survival responses in B cells. Consequently, mice displayed defective B cell maturation, antibody production, and formation of secondary lymphoid organs and tissues. Thus, IkappaBalpha nuclear export is essential to maintain constitutive, canonical, and noncanonical NF-kappaB activation potentials in mature B cells in vivo

Fanconi anemia (FA) is a rare familial genome instability syndrome caused by mutations in FA genes that results in defective DNA crosslink repair. Activation of the FA pathway requires the FA core ubiquitin ligase complex-dependent monoubiquitination of 2 interacting FA proteins, FANCI and FANCD2. Although loss of either FANCI or FANCD2 is known to prevent monoubiquitination of its respective partner, it is unclear whether FANCI has any additional domains that may be important in promoting DNA repair, independent of its monoubiquitination. Here, we focus on an FA-I patient-derived FANCI mutant protein, R1299X (deletion of 30 residues from its C-terminus), to characterize important structural region(s) in FANCI that is required to activate the FA pathway. We show that, within this short 30 amino acid stretch contains 2 separable functional signatures, a nuclear localization signal and a putative EDGE motif, that is critical for the ability of FANCI to properly monoubiquitinate FANCD2 and promote DNA crosslink resistance. Our study enable us to conclude that, although proper nuclear localization of FANCI is crucial for robust FANCD2 monoubiquitination, the putative FANCI EDGE motif is important for DNA crosslink repair

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

Activation of the Fanconi anemia (FA) DNA damage-response pathway results in the monoubiquitination of FANCD2, which is regulated by the nuclear FA core ubiquitin ligase complex. A FANCD2 protein sequence-based homology search facilitated the discovery of FANCI, a second monoubiquitinated component of the FA pathway. Biallelic mutations in the gene coding for this protein were found in cells from four FA patients, including an FA-I reference cell line.

The mechanisms involved in activation of the transcription factor NF-kappaB by genotoxic agents are not well understood. Previously, we provided evidence that a regulatory subunit of the IkappaB kinase (IKK) complex, NF-kappaB essential modulator (NEMO)/IKKgamma, is a component of a nuclear signal that is generated after DNA damage to mediate NF-kappaB activation. Here, we found that etoposide (VP16) and camptothecin (CPT) induced increases in intracellular free calcium levels at 60 minutes after stimulation of CEM T leukemic cells. Inhibition of calcium increases by calcium chelators, BAPTA-AM and EGTA-AM, abrogated NF-kappaB activation by these agents in several cell types examined. Conversely, thapsigargin and ionomycin attenuated the BAPTA-AM effects and promoted NF-kappaB activation by the genotoxic stimuli. Analyses of nuclear NEMO levels in VP16 treated cells suggested that calcium was required for nuclear export of NEMO. Inhibition of the nuclear exporter CRM1 by leptomycin B did not interfere with NEMO nuclear export. Similarly, deficiency of a plausible calcium-dependent nuclear-export receptor calreticulin failed to prevent NF-kappaB activation by VP16. However, temperature inactivation of the Ran guanine nucleotide exchange factor RCC1 in the tsBN2 cell line harboring a temperature sensitive mutant of RCC1 blocked NF-kappaB activation induced by genotoxic stimuli. Over-expression of Ran in this cell model showed that DNA damage stimuli induced formation of a complex between Ran and NEMO, suggesting that RCC1 regulated NF-kappaB activation through the modulation of RanGTP. Indeed, evidence for VP16-inducible interaction between Ran-GTP and NEMO could be obtained by means of GST-pull down assays using GST fused to the Ran binding domain of RanBP2, which specifically interacts with the GTP-bound form of Ran. BAPTA-AM did not alter these interactions suggesting that calcium is a necessary step beyond the formation of a Ran-GTP-NEMO complex in the nucleus. These results suggest that calcium has a unique role in genotoxic stress-induced NF-kappaB signaling by regulating nuclear export of NEMO subsequent to the formation of a nuclear export complex composed of Ran-GTP, NEMO and presumably an undefined nuclear export receptor