Centrin-2 is an evolutionarily conserved, calmodulin-related protein, which is involved in multiple cellular functions including centrosome regulation and nucleotide excision repair (NER) of DNA. Particularly to exert the latter function, complex formation with the XPC protein, the pivotal NER damage recognition factor, is crucial. Here, we show that the C-terminal half of centrin-2, containing two calcium-binding EF-hand motifs, is necessary and sufficient for both its localization to the centrosome and interaction with XPC. In XPC-deficient cells, nuclear localization of overexpressed centrin-2 largely depends on co-overexpression of XPC, and mutational analyses of the C-terminal domain suggest that XPC and the major binding partner in the centrosome share a common binding surface on the centrin-2 molecule. On the other hand, the N-terminal domain of centrin-2 also contains two EF-hand motifs but shows only low-binding affinity for calcium ions. Although the N-terminal domain is dispensable for enhancement of the DNA damage recognition activity of XPC, it contributes to augmenting rather weak physical interaction between XPC and XPA, another key factor involved in NER. These results suggest that centrin-2 may have evolved to bridge two protein factors, one with high affinity and the other with low affinity, thereby allowing delicate regulation of various biological processes.
Xeroderma pigmentosum group C (XPC) protein plays an essential role in DNA damage recognition in mammalian global genome nucleotide excision repair (NER). Here, we analyze the functional basis of NER inactivation caused by a single amino acid substitution (Trp to Ser at position 690) in XPC, previously identified in the XPC patient XP13PV. The Trp690Ser change dramatically affects the in vivo stability of the XPC protein, thereby causing a significant reduction of its steady-state level in XP13PV fibroblasts. Despite normal heterotrimeric complex formation and physical interactions with other NER factors, the mutant XPC protein lacks binding affinity for both undamaged and damaged DNA. Thus, this single amino acid substitution is sufficient to compromise XPC function through both quantitative and qualitative alterations of the protein. Although the mutant XPC fails to recognize damaged DNA, it is still capable of accumulating in a UV-damaged DNA-binding protein (UV-DDB)-dependent manner to UV-damaged subnuclear domains. However, the NER factors transcription factor IIH and XPA failed to colocalize stably with the mutant XPC. As well as highlighting the importance of UV-DDB in recruiting XPC to UV-damaged sites, these findings demonstrate the role of DNA binding by XPC in the assembly of subsequent NER intermediate complexes.
Abstract Non-homologous end-joining (NHEJ), which can promote genomic instability when dysfunctional, is a major DNA double-strand break (DSB) repair pathway. Although ubiquitylation of the core NHEJ factor, Ku (Ku70-Ku80), which senses broken DNA ends, is important for its removal from sites of damage upon completion of NHEJ, the mechanism regulating Ku ubiquitylation remains elusive. We provide evidence showing that the ubiquitin carboxyl-terminal hydrolase L3 (UCHL3) interacts with and directly deubiquitylates one of the Ku heterodimer subunits, Ku80. Additionally, depleting UCHL3 resulted in reduced Ku80 foci formation, Ku80 binding to chromatin after DSB induction, moderately sensitized cells to ionizing radiation and decreased NHEJ efficiencies. Mechanistically, we show that DNA damage induces UCHL3 phosphorylation, which is dependent on ATM, downstream NHEJ factors and UCHL3 catalytic activity. Furthermore, this phosphorylation destabilizes UCHL3, despite having no effect on its catalytic activity. Collectively, these data suggest that UCHL3 facilitates cellular viability after DSB induction by antagonizing Ku80 ubiquitylation to enhance Ku80 retention at sites of damage.
Abstract The nucleus of mammalian cells is compartmentalized by nuclear bodies such as nuclear speckles, however, involvement of nuclear bodies, especially nuclear speckles, in DNA repair has not been actively investigated. Here, our focused screen for nuclear speckle factors involved in homologous recombination (HR), which is a faithful DNA double-strand break (DSB) repair mechanism, identified transcription-related nuclear speckle factors as potential HR regulators. Among the top hits, we provide evidence showing that USP42, which is a hitherto unidentified nuclear speckles protein, promotes HR by facilitating BRCA1 recruitment to DSB sites and DNA-end resection. We further showed that USP42 localization to nuclear speckles is required for efficient HR. Furthermore, we established that USP42 interacts with DHX9, which possesses DNA–RNA helicase activity, and is required for efficient resolution of DSB-induced R-loop. In conclusion, our data propose a model in which USP42 facilitates BRCA1 loading to DSB sites, resolution of DSB-induced R-loop and preferential DSB repair by HR, indicating the importance of nuclear speckle-mediated regulation of DSB repair.
An accurate method using image sharpness to determine the best focusing is proposed for ultra-high-voltage electron microscopy. This method maximizes image sharpness for adjusting the focus. Five images with different defocus values are used to calculate the image sharpness. To obtain the best focus value that produces greatest image sharpness, fitting the quasi-Gaussian function to five image sharpness is a suitable alternative. This method, which maximizes image sharpness, gives better accuracy than the wobbler method for the ultra-high-voltage electron microscope. The focusing area can be selected without moving the field of view, because the focusing area can be selected at almost any area in the image.
