Enhanced mitochondrial fusion during a critical period of synaptic plasticity in adult-born neurons.

Integration of new neurons into adult hippocampal circuits is a process coordinated by local and long-range synaptic inputs. To achieve stable integration and uniquely contribute to hippocampal function, immature neurons are endowed with a critical period of heightened synaptic plasticity, yet it remains unclear which mechanisms sustain this form of plasticity during neuronal maturation. We found that as new neurons enter their critical period, a transient surge in fusion dynamics stabilizes elongated mitochondrial morphologies in dendrites to fuel synaptic plasticity. Conditional ablation of fusion dynamics to prevent mitochondrial elongation selectively impaired spine plasticity and synaptic potentiation, disrupting neuronal competition for stable circuit integration, ultimately leading to decreased survival. Despite profuse mitochondrial fragmentation, manipulation of competition dynamics was sufficient to restore neuronal survival but left neurons poorly responsive to experience at the circuit level. Thus, by enabling synaptic plasticity during the critical period, mitochondrial fusion facilitates circuit remodeling by adult-born neurons.

Exploring the burden of mixed dyslipidemia in patients with type 2 diabetes mellitus: A cross-sectional study in Kashmir, India.

BACKGROUND: Diabetes-related dyslipidemia is a multifaceted, complicated disorder characterized by an abnormal lipid profile in individuals with diabetes. The incidence of different types of dyslipidemia, however, was not a focus of prior investigations. The patients were characterized into three categories of dyslipidemia. Different patterns of dyslipidemia were combined into single dyslipidemia (7 patterns), mixed dyslipidemia (16 patterns), and triple dyslipidemia (4 patterns). METHODS: In this cross-sectional study, 586 people suffering from type 2 diabetes mellitus (T2DM) were included. We assessed the serum lipid profile and used log (TG/HDL-C) to determine the atherogenic index of plasma (AIP). Dyslipidemia was examined as a categorical variable, and the findings were presented as percentages and numbers. To compare categorical variables, we either utilized Fisher exact tests or Chi square tests. RESULTS: The study comprised of 586 T2DM patients, with 310 (52.9%) women and 276 (47.1%) men. Women have significantly higher hypertension (33.6%) as compared to men (23.2%). 18.94% (111) of patients were having coronary artery disease (CAD) history consisting of 12.28% (72) females and 6.66% (39) males, a difference which is statistically significant. 98.12% of total individuals had as a minimum of one lipid abnormality. 4.61% (27) of study subjects were having isolated dyslipidemia and 93.51% (548) had dual or triple pattern of dyslipidemia (mixed dyslipidemia). High AIP >0.24 (94.8%) was the most predominant trend of dyslipidemia. The dual combination of AIP (>0.24) and HDL (<50 mg/dL in Females and <40 mg/dL in Males) was found to be the most common pattern of mixed dyslipidemia (68.08%). The most prevalent trend of isolated dyslipidemia was found to be high AIP (>0.24), In patients with CAD history. Among the mixed dyslipidemia, the common pattern of dyslipidemia (71.17%) was the dual combination of high AIP (>0.24) and low HDL (<50 mg/dL women and <40 mg/dL males). The triple combination of TG (≥200 mg/dL) and HDL (<40 and <50 mg/dL) and LDL (≥100 mg/dL) was only found in females. CONCLUSION: In conclusion, dyslipidemia is highly prevalent in T2DM patients, with mixed dyslipidemia being the most common type observed in the community of Kashmir valley, India. High AIP was the most prevalent pattern in the current investigation.

Metabolic control of adult neural stem cell self-renewal by the mitochondrial protease YME1L.

The transition between quiescence and activation in neural stem and progenitor cells (NSPCs) is coupled with reversible changes in energy metabolism with key implications for lifelong NSPC self-renewal and neurogenesis. How this metabolic plasticity is ensured between NSPC activity states is unclear. We find that a state-specific rewiring of the mitochondrial proteome by the i-AAA peptidase YME1L is required to preserve NSPC self-renewal. YME1L controls the abundance of numerous mitochondrial substrates in quiescent NSPCs, and its deletion activates a differentiation program characterized by broad metabolic changes causing the irreversible shift away from a fatty-acid-oxidation-dependent state. Conditional Yme1l deletion in adult NSPCs in vivo results in defective self-renewal and premature differentiation, ultimately leading to NSPC pool depletion. Our results disclose an important role for YME1L in coordinating the switch between metabolic states of NSPCs and suggest that NSPC fate is regulated by compartmentalized changes in protein network dynamics.

Spironolactone-induced XPB degradation requires TFIIH integrity and ubiquitin-selective segregase VCP/p97.

Mineralocorticoid and androgen receptor antagonist, spironolactone, was recently identified as an inhibitor of nucleotide excision repair (NER), acting via induction of proteolysis of TFIIH component Xeroderma Pigmentosum B protein (XPB). This activity provides a strong rationale for repurposing spironolactone for cancer therapy. Here, we report that the spironolactone-induced XPB proteolysis is mediated through ubiquitin-selective segregase, valosin-containing protein (VCP)/p97. We show that spironolactone induces a dose- and time-dependent degradation of XPB but not XPD, and that the XPB degradation is blocked by VCP/p97 inhibitors DBeQ, NMS-873, and neddylation inhibitor MLN4924. Moreover, the cellular treatment by VCP/p97 inhibitors leads to the accumulation of ubiquitin conjugates of XPB but not XPD. VCP/p97 knockdown by inducible shRNA does not affect XPB level but compromises the spironolactone-induced XPB degradation. Also, VCP/p97 interacts with XPB upon treatment of spironolactone and proteasome inhibitor MG132, while the VCP/p97 adaptor UBXD7 binds XPB and its ubiquitin conjugates. Additionally, ATP analog-mediated inhibition of Cdk7 significantly decelerates spironolactone-induced XPB degradation. Likewise, engaging TFIIH to NER by UV irradiation slows down spironolactone-induced XPB degradation. These results indicate that the spironolactone-induced XPB proteolysis requires VCP/p97 function and that XPB within holo-TFIIH rather than core-TFIIH is more vulnerable to spironolactone-induced proteolysis. Abbreviations NER: nucleotide excision repair; TFIIH: transcription factor II H; CAK: Cdk-activating kinase (CAK) complex; XPB: Xeroderma Pigmentosum type B; VCP/p97: valosin-containing protein/p97; Cdk7: cyclin-dependent kinase 7; NAE: NEDD8-activating enzyme; IP: immunoprecipitation.

USP7-mediated deubiquitination differentially regulates CSB but not UVSSA upon UV radiation-induced DNA damage.

Cockayne syndrome group B (CSB) protein participates in transcription-coupled nucleotide excision repair. The stability of CSB is known to be regulated by ubiquitin-specific protease 7 (USP7). Yet, whether USP7 acts as a deubiquitinating enzyme for CSB is not clear. Here, we demonstrate that USP7 deubiquitinates CSB to maintain its levels after ultraviolet (UV)-induced DNA damage. While both CSB and UV-stimulated scaffold protein A (UVSSA) exhibit a biphasic decrease and recovery upon UV irradiation, only CSB recovery depends on USP7, which physically interacts with and deubiquitinates CSB. Meanwhile, CSB overexpression stabilizes UVSSA, but decrease UVSSA's presence in nuclease-releasable/soluble chromatin, and increase the presence of ubiquitinated UVSSA in insoluble chromatin alongside CSB-ubiquitin conjugates. Remarkably, CSB overexpression also decreases CSB association with USP7 and UVSSA in soluble chromatin. UVSSA exists in several ubiquitinated forms, of which mono-ubiquitinated form and other ubiquitinated UVSSA forms are detectable upon 6xHistidine tag-based purification. The ubiquitinated UVSSA forms, however, are not cleavable by USP7 in vitro. Furthermore, USP7 disruption does not affect RNA synthesis but decreases the recovery of RNA synthesis following UV exposure. These results reveal a role of USP7 as a CSB deubiquitinating enzyme for fine-tuning the process of TC-NER in human cells.

Loss of the mitochondrial i-AAA protease YME1L leads to ocular dysfunction and spinal axonopathy.

Disturbances in the morphology and function of mitochondria cause neurological diseases, which can affect the central and peripheral nervous system. The i-AAA protease YME1L ensures mitochondrial proteostasis and regulates mitochondrial dynamics by processing of the dynamin-like GTPase OPA1. Mutations in YME1L cause a multi-systemic mitochondriopathy associated with neurological dysfunction and mitochondrial fragmentation but pathogenic mechanisms remained enigmatic. Here, we report on striking cell-type-specific defects in mice lacking YME1L in the nervous system. YME1L-deficient mice manifest ocular dysfunction with microphthalmia and cataracts and develop deficiencies in locomotor activity due to specific degeneration of spinal cord axons, which relay proprioceptive signals from the hind limbs to the cerebellum. Mitochondrial fragmentation occurs throughout the nervous system and does not correlate with the degenerative phenotype. Deletion of Oma1 restores tubular mitochondria but deteriorates axonal degeneration in the absence of YME1L, demonstrating that impaired mitochondrial proteostasis rather than mitochondrial fragmentation causes the observed neurological defects.

Human CRL4DDB2 ubiquitin ligase preferentially regulates post-repair chromatin restoration of H3K56Ac through recruitment of histone chaperon CAF-1.

Acetylated histone H3 lysine 56 (H3K56Ac) diminishes in response to DNA damage but is restored following DNA repair. Here, we report that CRL4DDB2 ubiquitin ligase preferentially regulates post-repair chromatin restoration of H3K56Ac through recruitment of histone chaperon CAF-1. We show that H3K56Ac accumulates at DNA damage sites. The restoration of H3K56Ac but not H3K27Ac, H3K18Ac and H3K14Ac depends on CAF-1 function, whereas all these acetylations are mediated by CBP/p300. The CRL4DDB2 components, DDB1, DDB2 and CUL4A, are also required for maintaining the H3K56Ac and H3K9Ac level in chromatin, and for restoring H3K56Ac following induction of DNA photolesions and strand breaks. Depletion of CUL4A decreases the recruitment of CAF-1 p60 and p150 to ultraviolet radiation- and phleomycin-induced DNA damage. Neddylation inhibition renders CRL4DDB2 inactive, decreases H3K56Ac level, diminishes CAF-1 recruitment and prevents H3K56Ac restoration. Mutation in the PIP box of DDB2 compromises its capability to elevate the H3K56Ac level but does not affect XPC ubiquitination. These results demonstrated a function of CRL4DDB2 in differential regulation of histone acetylation in response to DNA damage, suggesting a novel role of CRL4DDB2 in repair-driven chromatin assembly.

UV radiation-induced SUMOylation of DDB2 regulates nucleotide excision repair.

Subunit 2 of DNA damage-binding protein complex (DDB2) is an early sensor of nucleotide excision repair (NER) pathway for eliminating DNA damage induced by UV radiation (UVR) and cisplatin treatments of mammalian cells. DDB2 is modified by ubiquitin and poly(ADP-ribose) (PAR) in response to UVR, and these modifications play a crucial role in regulating NER. Here, using immuno-analysis of irradiated cell extracts, we have identified multiple post-irradiation modifications of DDB2 protein. Interestingly, although the DNA lesions induced by both UVR and cisplatin are corrected by NER, only the UV irradiation, but not the cisplatin treatment, induces any discernable DDB2 modifications. We, for the first time, show that the appearance of UVR-induced DDB2 modifications depend on the binding of DDB2 to the damaged chromatin and the participation of functionally active 26S proteasome. The in vitro and in vivo analysis revealed that SUMO-1 conjugations comprise a significant portion of these UVR-induced DDB2 modifications. Mapping of SUMO-modified sites demonstrated that UVR-induced SUMOylation occurs on Lys-309 residue of DDB2 protein. Mutation of Lys-309 to Arg-309 diminished the DDB2 SUMOylation observable both in vitro and in vivo. Moreover, K309R mutated DDB2 lost its function of recruiting XPC to the DNA damage sites, as well as the ability to repair cyclobutane pyrimidine dimers following cellular UV irradiation. Taken together, our results indicate that DDB2 is modified by SUMOylation upon UV irradiation, and this post-translational modification plays an important role in the initial recognition and processing of UVR-induced DNA damage occurring within the context of chromatin.

UV-induced proteolysis of RNA polymerase II is mediated by VCP/p97 segregase and timely orchestration by Cockayne syndrome B protein.

RNA polymerase II (RNAPII) acts as a damage sensor for transcription-coupled nucleotide excision repair (TC-NER) and undergoes proteolytic clearance from damaged chromatin by the ubiquitin-proteasome system (UPS). Here, we report that Valosin-containing protein (VCP)/p97, a druggable oncotarget, is essential for RNAPII's proteolytic clearance in mammalian cells. We show that inhibition of VCP/p97, or siRNA-mediated ablation of VCP/p97 and its cofactors UFD1 and UBXD7 severely impairs ultraviolet radiation (UVR)-induced RNAPII degradation. VCP/p97 interacts with RNAPII, and the interaction is enhanced by Cockayne syndrome B protein (CSB). However, the VCP/p97-mediated RNAPII proteolysis occurs independent of CSB. Surprisingly, CSB enhances UVR-induced RNAPII ubiquitination but delays its turnover. Additionally, VCP/p97-mediated RNAPII turnover occurs with and without Von Hippel-Lindau tumor suppressor protein (pVHL), a known substrate receptor of Elongin E3 ubiquitin ligase for RNAPII. Moreover, pVHL re-expression improves cell viability following UVR. Whereas, VCP/p97 inhibition decreases cell viability and enhances a low-dose UVR killing in presence of pVHL. These findings reveal a function of VCP/p97 segregase in UVR-induced RNAPII degradation in mammalian cells, and suggest a role of CSB in coordinating VCP/p97-mediated extraction of ubiquitinated RNAPII and CSB itself from chromatin.

ATR- and ATM-Mediated DNA Damage Response Is Dependent on Excision Repair Assembly during G1 but Not in S Phase of Cell Cycle.

Cell cycle checkpoint is mediated by ATR and ATM kinases, as a prompt early response to a variety of DNA insults, and culminates in a highly orchestrated signal transduction cascade. Previously, we defined the regulatory role of nucleotide excision repair (NER) factors, DDB2 and XPC, in checkpoint and ATR/ATM-dependent repair pathway via ATR and ATM phosphorylation and recruitment to ultraviolet radiation (UVR)-induced damage sites. Here, we have dissected the molecular mechanisms of DDB2- and XPC- mediated regulation of ATR and ATM recruitment and activation upon UVR exposures. We show that the ATR and ATM activation and accumulation to UVR-induced damage not only depends on DDB2 and XPC, but also on the NER protein XPA, suggesting that the assembly of an active NER complex is essential for ATR and ATM recruitment. ATR and ATM localization and H2AX phosphorylation at the lesion sites occur as early as ten minutes in asynchronous as well as G1 arrested cells, showing that repair and checkpoint-mediated by ATR and ATM starts early upon UV irradiation. Moreover, our results demonstrated that ATR and ATM recruitment and H2AX phosphorylation are dependent on NER proteins in G1 phase, but not in S phase. We reasoned that in G1 the UVR-induced ssDNA gaps or processed ssDNA, and the bound NER complex promote ATR and ATM recruitment. In S phase, when the UV lesions result in stalled replication forks with long single-stranded DNA, ATR and ATM recruitment to these sites is regulated by different sets of proteins. Taken together, these results provide evidence that UVR-induced ATR and ATM recruitment and activation differ in G1 and S phases due to the existence of distinct types of DNA lesions, which promote assembly of different proteins involved in the process of DNA repair and checkpoint activation.

Valosin-containing Protein (VCP)/p97 Segregase Mediates Proteolytic Processing of Cockayne Syndrome Group B (CSB) in Damaged Chromatin.

Cockayne syndrome group A and B (CSB) proteins act in transcription-coupled repair, a subpathway of nucleotide excision repair. Here we demonstrate that valosin-containing protein (VCP)/p97 segregase functions in ultraviolet radiation (UVR)-induced ubiquitin-mediated CSB degradation. We show that VCP/p97 inhibition and siRNA-mediated ablation of VCP/p97 and its cofactors UFD1 and UBXD7 impair CSB degradation. VCP/p97 inhibition also results in the accumulation of CSB in chromatin. Moreover, VCP/p97 interacts with both native and ubiquitin-conjugated forms of CSB. The localized cellular UVR exposures lead to VCP/p97 accumulation at DNA damage spots, forming distinct UVR-induced foci. However, manifestation of VCP/p97 foci is independent of CSB and UBXD7. Furthermore, VCP/p97 and UBXD7 associate with the Cockayne syndrome group A-DDB1-Cul4A complex, an E3 ligase responsible for CSB ubiquitination. Compromising proteasome and VCP/p97 function allows accumulation of both native and ubiquitinated CSB and results in an increase of UBXD7, proteasomal RPN2, and Sug1 in the chromatin compartment. Surprisingly, both biochemical inhibition and genetic defect of VCP/p97 enhance the recovery of RNA synthesis following UVR, whereas both VCP/p97 and proteasome inhibitions decrease cell viability. Our findings reveal a new role of VCP/p97 segregase in the timely processing of ubiquitinated CSB from damaged chromatin.

Damaged DNA-binding protein down-regulates epigenetic mark H3K56Ac through histone deacetylase 1 and 2.

Acetylated histone H3 lysine 56 (H3K56Ac) is one of the reversible histone post-translational modifications (PTMs) responsive to DNA damage. We previously described a biphasic decrease and increase of epigenetic mark H3K56Ac in response to ultraviolet radiation (UVR)-induced DNA damage. Here, we report a new function of UV damaged DNA-binding protein (DDB) in deacetylation of H3K56Ac through specific histone deacetylases (HDACs). We show that simultaneous depletion of HDAC1/2 compromises the deacetylation of H3K56Ac, while depletion of HDAC1 or HDAC2 alone has no effect on H3K56Ac. The H3K56Ac deacetylation does not require functional nucleotide excision repair (NER) factors XPA and XPC, but depends on the function of upstream factors DDB1 and DDB2. UVR enhances the association of DDB2 with HDAC1 and, enforced DDB2 expression leads to translocation of HDAC1 to UVR-damaged chromatin. HDAC1 and HDAC2 are recruited to UVR-induced DNA damage spots, which are visualized by anti-XPC immunofluorescence. Dual HDAC1/2 depletion decreases XPC ubiquitination, but does not affect the recruitment of DDB2 to DNA damage. By contrast, the local accumulation of γH2AX at UVR-induced DNA damage spots was compromised upon HDAC1 as well as dual HDAC1/2 depletions. Additionally, UVR-induced ATM activation decreased in H12899 cells expressing H3K56Ac-mimicing H3K56Q. These results revealed a novel role of DDB in H3K56Ac deacetylation during early step of NER and the existence of active functional cross-talk between DDB-mediated damage recognition and H3K56Ac deacetylation.

USP7 deubiquitinase promotes ubiquitin-dependent DNA damage signaling by stabilizing RNF168.

During DNA damage response (DDR), histone ubiquitination by RNF168 is a critical event, which orchestrates the recruitment of downstream DDR factors, e.g. BRCA1 and 53BP1. Here, we report USP7 deubiquitinase regulates the stability of RNF168. We showed that USP7 disruption impairs H2A and ultraviolet radiation (UVR)-induced γH2AX monoubiquitination, and decreases the levels of pBmi1, Bmi1, RNF168 and BRCA1. The effect of USP7 disruption was recapitulated by siRNA-mediated USP7 depletion. The USP7 disruption also compromises the formation of UVR-induced foci (UVRIF) and ionizing radiation-induced foci (IRIF) of monoubiquitinated H2A (uH2A) and polyubiquitinated H2AX/A, and subsequently affects UVRIF and IRIF of BRCA1 as well as the IRIF of 53BP1. USP7 was shown to physically bind RNF168 in vitro and in vivo. Overexpression of wild-type USP7, but not its interaction-defective mutant, prevents UVR-induced RNF168 degradation. The USP7 mutant is unable to cleave Ub-conjugates of RNF168 in vivo. Importantly, ectopic expression of RNF168, or both RNF8 and RNF168 together in USP7-disrupted cells, significantly rescue the formation of UVRIF and IRIF of polyubiquitinated H2A and BRCA1. Taken together, these findings reveal an important role of USP7 in regulating ubiquitin-dependent signaling via stabilization of RNF168.

Cdt2-mediated XPG degradation promotes gap-filling DNA synthesis in nucleotide excision repair.

Xeroderma pigmentosum group G (XPG) protein is a structure-specific repair endonuclease, which cleaves DNA strands on the 3' side of the DNA damage during nucleotide excision repair (NER). XPG also plays a crucial role in initiating DNA repair synthesis through recruitment of PCNA to the repair sites. However, the fate of XPG protein subsequent to the excision of DNA damage has remained unresolved. Here, we show that XPG, following its action on bulky lesions resulting from exposures to UV irradiation and cisplatin, is subjected to proteasome-mediated proteolytic degradation. Productive NER processing is required for XPG degradation as both UV and cisplatin treatment-induced XPG degradation is compromised in NER-deficient XP-A, XP-B, XP-C, and XP-F cells. In addition, the NER-related XPG degradation requires Cdt2, a component of an E3 ubiquitin ligase, CRL4(Cdt2). Micropore local UV irradiation and in situ Proximity Ligation assays demonstrated that Cdt2 is recruited to the UV-damage sites and interacts with XPG in the presence of PCNA. Importantly, Cdt2-mediated XPG degradation is crucial to the subsequent recruitment of DNA polymerase δ and DNA repair synthesis. Collectively, our data support the idea of PCNA recruitment to damage sites which occurs in conjunction with XPG, recognition of the PCNA-bound XPG by CRL4(Cdt2) for specific ubiquitylation and finally the protein degradation. In essence, XPG elimination from DNA damage sites clears the chromatin space needed for the subsequent recruitment of DNA polymerase δ to the damage site and completion of gap-filling DNA synthesis during the final stage of NER.

Ubiquitin-specific protease 7 regulates nucleotide excision repair through deubiquitinating XPC protein and preventing XPC protein from undergoing ultraviolet light-induced and VCP/p97 protein-regulated proteolysis.

Ubiquitin specific protease 7 (USP7) is a known deubiquitinating enzyme for tumor suppressor p53 and its downstream regulator, E3 ubiquitin ligase Mdm2. Here we report that USP7 regulates nucleotide excision repair (NER) via deubiquitinating xeroderma pigmentosum complementation group C (XPC) protein, a critical damage recognition factor that binds to helix-distorting DNA lesions and initiates NER. XPC is ubiquitinated during the early stage of NER of UV light-induced DNA lesions. We demonstrate that transiently compromising cellular USP7 by siRNA and chemical inhibition leads to accumulation of ubiquitinated forms of XPC, whereas complete USP7 deficiency leads to rapid ubiquitin-mediated XPC degradation upon UV irradiation. We show that USP7 physically interacts with XPC in vitro and in vivo. Overexpression of wild-type USP7, but not its catalytically inactive or interaction-defective mutants, reduces the ubiquitinated forms of XPC. Importantly, USP7 efficiently deubiquitinates XPC-ubiquitin conjugates in deubiquitination assays in vitro. We further show that valosin-containing protein (VCP)/p97 is involved in UV light-induced XPC degradation in USP7-deficient cells. VCP/p97 is readily recruited to DNA damage sites and colocalizes with XPC. Chemical inhibition of the activity of VCP/p97 ATPase causes an increase in ubiquitinated XPC on DNA-damaged chromatin. Moreover, USP7 deficiency severely impairs the repair of cyclobutane pyrimidine dimers and, to a lesser extent, affects the repair of 6-4 photoproducts. Taken together, our findings uncovered an important role of USP7 in regulating NER via deubiquitinating XPC and by preventing its VCP/p97-regulated proteolysis.

USP3 counteracts RNF168 via deubiquitinating H2A and γH2AX at lysine 13 and 15.

Histone ubiquitination plays a vital role in DNA damage response (DDR), which is important for maintaining genomic integrity in eukaryotic cells. In DDR, ubiquitination of histone H2A and γH2AX by the concerted action of ubiquitin (Ub) ligases, RNF168 and RNF8, generates a cascade of ubiquitination signaling. However, little is known about deubiquitinating enzymes (DUBs) that may catalyze the removal of Ub from these histones. This study demonstrated that USP3, an apparent DUB for mono-ubiquitinated H2A, is indeed the enzyme for deubiquitinating Ub conjugates of γH2AX and H2A from lysine sites, where the ubiquitination is initiated by RNF168. Here, we showed that ectopic expression of USP3 led to the deubiquitination of both H2A and γH2AX in response to UV-induced DNA damage. Moreover, ectopic USP3 expression abrogated FK2 antibody-reactive Ub-conjugate foci, which co-localize with damage-induced γH2AX foci. In addition, USP3 overexpression impaired the accumulation of downstream repair factors BRCA1 and 53BP1 at the damage sites in response to both UV and γ-irradiation. We further identified that the USP3 removes Ub at lysine 13 and 15 of H2A and γH2AX, as well as lysine 118 and 119 of H2AX in response to DNA damage. Taken together, the results suggested that USP3 is a negative regulator of ubiquitination signaling, counteracting RNF168- and RNF8-mediated ubiquitination.

NER initiation factors, DDB2 and XPC, regulate UV radiation response by recruiting ATR and ATM kinases to DNA damage sites.

ATR and ATM kinases are central to the checkpoint activation in response to DNA damage and replication stress. However, the nature of the signal, which initially activates these kinases in response to UV damage, is unclear. Here, we have shown that DDB2 and XPC, two early UV damage recognition factors, are required for the damage-specific ATR and ATM recruitment and phosphorylation. ATR and ATM physically interacted with XPC and promptly localized to the UV damage sites. ATR and ATM recruitment and their phosphorylation were negatively affected in cells defective in DDB2 or XPC functions. Consequently, the phosphorylation of ATR and ATM substrates, Chk1, Chk2, H2AX, and BRCA1 was significantly reduced or abrogated in mutant cells. Furthermore, UV exposure of cells defective in DDB2 or XPC resulted in a marked decrease in BRCA1 and Rad51 recruitment to the damage site. Conversely, ATR- and ATM-deficiency failed to affect the recruitment of DDB2 and XPC to the damage site, and therefore did not influence the NER efficiency. These findings demonstrate a novel function of DDB2 and XPC in maintaining a vital cross-talk with checkpoint proteins, and thereby coordinating subsequent repair and checkpoint activation.

p38 MAPK- and Akt-mediated p300 phosphorylation regulates its degradation to facilitate nucleotide excision repair.

Besides the primary histone acetyltransferase (HAT)-mediated chromatin remodeling function, co-transcriptional factor, p300, is also known to play a distinct role in DNA repair. However, the exact mechanism of p300 function in DNA repair has remained unclear and difficult to discern due to the phosphorylation and degradation of p300 in response to DNA damage. Here, we have demonstrated that p300 is only degraded in the presence of specific DNA lesions, which are the substrates of nucleotide excision repair (NER) pathway. In contrast, DNA double-strand breaks fail to degrade p300. Degradation is initiated by phosphorylation of p300 at serine 1834, which is catalyzed by the cooperative action of p38 mitogen-activated protein kinases and Akt kinases. In depth, functional analysis revealed that (i) p300 and CBP act redundantly in repairing ultraviolet (UV) lesions, (ii) the phosphorylation of p300 at S1834 is critical for efficient removal of UV-induced cyclobutane pyrimidine dimers and (iii) p300 is recruited to DNA damage sites located within heterochromatin. Taken together, we conclude that phosphorylated p300 initially acetylates histones to relax heterochromatin to allow damage recognition factors access to damage DNA. Thereupon, p300 is promptly degraded to allow the sequential recruitment of downstream repair proteins for successful execution of NER.

Lack of CAK complex accumulation at DNA damage sites in XP-B and XP-B/CS fibroblasts reveals differential regulation of CAK anchoring to core TFIIH by XPB and XPD helicases during nucleotide excision repair.

Transcription factor II H (TFIIH) is composed of core TFIIH and Cdk-activating kinase (CAK) complexes. Besides transcription, TFIIH also participates in nucleotide excision repair (NER), verifying DNA lesions through its helicase components XPB and XPD. The assembly state of TFIIH is known to be affected by truncation mutations in xeroderma pigmentosum group G/Cockayne syndrome (XP-G/CS). Here, we showed that CAK component MAT1 was rapidly recruited to UV-induced DNA damage sites, co-localizing with core TFIIH component p62, and dispersed from the damage sites upon completion of DNA repair. While the core TFIIH-CAK association remained intact, MAT1 failed to accumulate at DNA damage sites in fibroblasts harboring XP-B or XP-B/CS mutations. Nevertheless, MAT1, XPD and XPC as well as XPG were able to accumulate at damage sites in XP-D fibroblasts, in which the core TFIIH-CAK association also remained intact. Interestingly, XPG recruitment was impaired in XP-B/CS fibroblasts derived from patients with mild phenotype, but persisted in XP-B/CS fibroblasts from severely affected patients resulting in a nonfunctional preincision complex. An examination of steady-state levels of RNA polymerase II (RNAPII) indicated that UV-induced RNAPII phosphorylation was dramatically reduced in XP-B/CS fibroblasts. These results demonstrated that the CAK rapidly disassociates from the core TFIIH upon assembly of nonfunctional preincision complex in XP-B and XP-B/CS cells. The persistency of nonfunctional preincision complex correlates with the severity exhibited by XP-B patients. The results suggest that XPB and XPD helicases differentially regulate the anchoring of CAK to core TFIIH during damage verification step of NER.