Caspase-Mediated Cleavage of the Transcription Factor Sp3: Possible Relevance to Cancer and the Lytic Cycle of Kaposi’s Sarcoma-Associated Herpesvirus

The ORF50 protein of Kaposi’s sarcoma-associated herpesvirus (KSHV) is the key viral protein that controls the switch from latency to lytic reactivation. It is a potent transactivator that can activate target gene promoters via interacting with other cellular DNA-binding transcription factors, such as Sp3.

N4-acetyldeoxycytosine DNA modification marks euchromatin regions in Arabidopsis thaliana

Background Direct analogs of chemically modified bases that carry important epigenetic information, such as 5-methylcytosine (m5C)/5-methyldeoxycytosine (5mC), 5-hydroxymethylcytosine (hm5C)/5-hydroxymethyldeoxycytosine (5hmC), and N6-methyladenosine (m6A)/N6-methyldeoxyadenosine (6mA), are detected in both RNA and DNA, respectively. The modified base N4-acetylcytosine (ac4C) is well studied in RNAs, but its presence and epigenetic roles in cellular DNA have not been explored. Results Here, we demonstrate the existence of N4-acetyldeoxycytosine (4acC) in genomic DNA of Arabidopsis with multiple detection methods. Genome-wide profiling of 4acC modification reveals that 4acC peaks are mostly distributed in euchromatin regions and present in nearly half of the expressed protein-coding genes in Arabidopsis. 4acC is mainly located around transcription start sites and positively correlates with gene expression levels. Imbalance of 5mC does not directly affect 4acC modification. We also characterize the associations of 4acC with 5mC and histone modifications that cooperatively regulate gene expression. Moreover, 4acC is also detected in genomic DNA of rice, maize, mouse, and human by mass spectrometry. Conclusions Our findings reveal 4acC as a hitherto unknown DNA modification in higher eukaryotes. We identify potential interactions of this mark with other epigenetic marks in gene expression regulation.

Intrinsic Strand-Incision Activity of Human UNG: Implications for Nick Generation in Immunoglobulin Gene Diversification

Uracil arises in cellular DNA by cytosine (C) deamination and erroneous replicative incorporation of deoxyuridine monophosphate opposite adenine. The former generates C → thymine transition mutations if uracil is not removed by uracil-DNA glycosylase (UDG) and replaced by C by the base excision repair (BER) pathway. The primary human UDG is hUNG. During immunoglobulin gene diversification in activated B cells, targeted cytosine deamination by activation-induced cytidine deaminase followed by uracil excision by hUNG is important for class switch recombination (CSR) and somatic hypermutation by providing the substrate for DNA double-strand breaks and mutagenesis, respectively. However, considerable uncertainty remains regarding the mechanisms leading to DNA incision following uracil excision: based on the general BER scheme, apurinic/apyrimidinic (AP) endonuclease (APE1 and/or APE2) is believed to generate the strand break by incising the AP site generated by hUNG. We report here that hUNG may incise the DNA backbone subsequent to uracil excision resulting in a 3´-α,β-unsaturated aldehyde designated uracil-DNA incision product (UIP), and a 5´-phosphate. The formation of UIP accords with an elimination (E2) reaction where deprotonation of C2´ occurs via the formation of a C1´ enolate intermediate. UIP is removed from the 3´-end by hAPE1. This shows that the first two steps in uracil BER can be performed by hUNG, which might explain the significant residual CSR activity in cells deficient in APE1 and APE2.

Bombyx mori Nucleopolyhedrovirus (BmNPV) Induces G2/M Arrest to Promote Viral Multiplication by Depleting BmCDK1

Understanding virus–host interaction is very important for delineating the mechanism involved in viral replication and host resistance. Baculovirus, an insect virus, can cause S or G2/M phase arrest in insect cells. However, the roles and mechanism of Baculovirus-mediated S or G2/M phase arrest are not fully understood. Our results, obtained using flow cytometry (FCM), tubulin-labeling, BrdU-labeling, and CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS), showed that Bombyx mori nucleopolyhedrovirus (BmNPV) induced G2/M phase arrest and inhibited cellular DNA replication as well as cell proliferation in BmN-SWU1 cells. We found that BmNPV induced G2/M arrest to support its replication and proliferation by reducing the expression of BmCDK1 and BmCyclin B. Co-immunoprecipitation assays confirmed that BmNPV IAP1 interacted with BmCDK1. BmNPV iap1 was involved in the process of BmNPV-induced G2/M arrest by reducing the content of BmCDK1. Taken together, our results improve the understanding of the virus–host interaction network, and provide a potential target gene that connects apoptosis and the cell cycle.

Tools for Decoding Ubiquitin Signaling in DNA Repair

The maintenance of genome stability requires dedicated DNA repair processes and pathways that are essential for the faithful duplication and propagation of chromosomes. These DNA repair mechanisms counteract the potentially deleterious impact of the frequent genotoxic challenges faced by cells from both exogenous and endogenous agents. Intrinsic to these mechanisms, cells have an arsenal of protein factors that can be utilised to promote repair processes in response to DNA lesions. Orchestration of the protein factors within the various cellular DNA repair pathways is performed, in part, by post-translational modifications, such as phosphorylation, ubiquitin, SUMO and other ubiquitin-like modifiers (UBLs). In this review, we firstly explore recent advances in the tools for identifying factors involved in both DNA repair and ubiquitin signaling pathways. We then expand on this by evaluating the growing repertoire of proteomic, biochemical and structural techniques available to further understand the mechanistic basis by which these complex modifications regulate DNA repair. Together, we provide a snapshot of the range of methods now available to investigate and decode how ubiquitin signaling can promote DNA repair and maintain genome stability in mammalian cells.

The Promotion of Genomic Instability in Human Fibroblasts by Adenovirus 12 Early Region 1B 55K Protein in the Absence of Viral Infection

The adenovirus 12 early region 1B55K (Ad12E1B55K) protein has long been known to cause non-random damage to chromosomes 1 and 17 in human cells. These sites, referred to as Ad12 modification sites, have marked similarities to classic fragile sites. In the present report we have investigated the effects of Ad12E1B55K on the cellular DNA damage response and on DNA replication, considering our increased understanding of the pathways involved. We have compared human skin fibroblasts expressing Ad12E1B55K (55K+HSF), but no other viral proteins, with the parental cells. Appreciable chromosomal damage was observed in 55K+HSFs compared to parental cells. Similarly, an increased number of micronuclei was observed in 55K+HSFs, both in cycling cells and after DNA damage. We compared DNA replication in the two cell populations; 55K+HSFs showed increased fork stalling and a decrease in fork speed. When replication stress was introduced with hydroxyurea the percentage of stalled forks and replication speeds were broadly similar, but efficiency of fork restart was significantly reduced in 55K+HSFs. After DNA damage, appreciably more foci were formed in 55K+HSFs up to 48 h post treatment. In addition, phosphorylation of ATM substrates was greater in Ad12E1B55K-expressing cells following DNA damage. Following DNA damage, 55K+HSFs showed an inability to arrest in cell cycle, probably due to the association of Ad12E1B55K with p53. To confirm that Ad12E1B55K was targeting components of the double-strand break repair pathways, co-immunoprecipitation experiments were performed which showed an association of the viral protein with ATM, MRE11, NBS1, DNA-PK, BLM, TOPBP1 and p53, as well as with components of the replisome, MCM3, MCM7, ORC1, DNA polymerase δ, TICRR and cdc45, which may account for some of the observed effects on DNA replication. We conclude that Ad12E1B55K impacts the cellular DNA damage response pathways and the replisome at multiple points through protein–protein interactions, causing genomic instability.

The base-editing enzyme APOBEC3A catalyzes cytosine deamination in RNA with low proficiency and high selectivity

Human APOBEC3A (A3A) is a nucleic acid-modifying enzyme that belongs to the cytidine deaminase family. Canonically, A3A catalyzes the deamination of cytosine into uracil in single-stranded DNA, an activity that makes A3A both a critical antiviral defense factor and a useful tool for targeted genome editing. However, off-target mutagenesis by A3A has been readily detected in both cellular DNA and RNA, which has been shown to promote oncogenesis. Given the importance of substrate discrimination for the physiological, pathological, and biotechnological activities of A3A, here we explore the mechanistic basis for its preferential targeting of DNA over RNA. Using a chimeric substrate containing a target ribocytidine within an otherwise DNA backbone, we demonstrate that a single hydroxyl at the sugar of the target base acts as a major selectivity determinant for deamination. To assess the contribution of bases neighboring the target cytosine, we show that overall RNA deamination is greatly reduced relative to that of DNA, but can be observed when ideal features are present, such as preferred sequence context and secondary structure. A strong dependence on idealized substrate features can also be observed with a mutant of A3A (eA3A, N57G) which has been employed for genome editing due to altered selectivity for DNA over RNA. Altogether, our work reveals a relationship between the overall decreased reactivity of A3A and increased substrate selectivity, and our results hold implications both for characterizing off-target mutagenesis and for engineering optimized DNA deaminases for base-editing technologies.

Evaluation of the Anticancer Potential of Crude, Irradiated Cerastes cerastes Snake Venom and Propolis Ethanolic Extract & Related Biological Alterations

We aimed to evaluate the anticancer potential of crude venom (CV), γ irradiated Certastes cerastes venom (IRRV), and propolis ethanolic extract (PEE). IRRV showed a higher toxicity than CV, while CV-PEE showed higher toxicity than IRRV and CV against lung [A549] and prostate [PC3] cancer cells. Toxicity to [A549] and [PC3] cells was concentration and cell type dependent. In comparison to controls, apoptotic genes showed a significant upregulation of P53 and Casp-3 and a downregulation of Bcl-2. Also, induced elevated DNA accumulation in the [S] phase post PC3 cell treatment with IRRV and CV, as well as a significant DNA accumulation at G2/M phase after IRRV treatment of A549 cells. In contrast, PC3 cells showed a negligible cellular DNA accumulation after PEE treatment. Glutathione reductase [GR] was reduced in case of PC3 and A549 cell treated with IRRV, CV, and PEE compared with its values in untreated cell control. The Malondialdehyde [MDA] values in both cells recorded a significant elevation post IRRV treatment compared to the rest of the treatment regimen and untreated cell control. Similarly, IRRV and CV-PEE mix showed obviously higher reactive oxygen species [ROS] values than PC3 and A549 cell treatments with CV and PEE.

Urine Cellular DNA Point Mutation and Methylation as Potential Biomarkers for the Detection of Urothelial Carcinoma

BackgroundPreviously, our team identified a seven-gene mutation panel in urine sediment to discriminate UBC from benign urological diseases. In the present study, we aimed to validate the panel in an expanded and close to natural population cohort of hematuria. Also, we tried to optimize the panel by incorporating methylation biomarkers. We performed external validation to investigate the robustness and stability of the novel panel.MethodsPatients with urothelial carcinomas and controls were prospectively recruited in clinical trial ChiCTR2000029980. The mutation panel was validated in the expanded cohort(n=333) from Hunan multicenter. Several UBC-specific methylation biomarkers were identified by comprehensive analyses of a series of TCGA, GEO and an independent cohorts, and examined in the expanded cohort. Random Forest algorithm was used to construct an optimal panel. External validation of the optimal panel was carried out in Beijing single center cohort(n=89). NGS technique was used to analyze the DNA point mutations and MS-PCR for methylation.ResultsThe AUC, sensitivity and specificity of the mutation panel in expanded cohort were 0.81, 0.67 and 0.90, respectively. After screening, only cg16966315, cg17945976 and cg24720571 were left for further analysis. The optimal panel consisted of cg24720571 and 8 point mutations, including TERT.chr5_1295228 G_A (TERT 228), FGFR3.chr4_1803568 C_G (FGFR3 568), TERT.chr5_1295250 G_A (TERT 250), FGFR3.chr4_1806099 A_G (FGFR3 099), PIK3CA.chr3_178936091 G_A (PIK3CA 091), PIK3CA.chr3_178952085 A_G (PIK3CA 085), PIK3CA.chr3_178936082 G_A (PIK3CA 082), HRAS.chr11_533874 T_C (HRAS 874). The AUC, sensitivity and specificity of the optimal panel in training group were 0.89, 0.84 and 0.79, respectively, and in test group were 0.95, 0.91 and 0.95, respectively. In the external validation, the AUC, sensitivity and specificity were 0.98, 0.93 and 0.93, respectively.ConclusionsThe optimal panel was obviously superior to previous mutation panel and showed a highly specific and robust performance. The optimal panel may be used as a replaceable approach for early detection of UC.Trial registrationThis research was registered in Chinese Clinical Trial Registry(ChiCTR2000029980).