Human SHPRH suppresses genomic instability through proliferating cell nuclear antigen polyubiquitination

Differential modifications of proliferating cell nuclear antigen (PCNA) determine DNA repair pathways at stalled replication forks. In yeast, PCNA monoubiquitination by the ubiquitin ligase (E3) yRad18 promotes translesion synthesis (TLS), whereas the lysine-63–linked polyubiquitination of PCNA by yRad5 (E3) promotes the error-free mode of bypass. The yRad5-dependent pathway is important to prevent genomic instability during replication, although its exact molecular mechanism is poorly understood. This mechanism has remained totally elusive in mammals because of the lack of apparent RAD5 homologues. We report that a putative tumor suppressor gene, SHPRH, is a human orthologue of yeast RAD5. SHPRH associates with PCNA, RAD18, and the ubiquitin-conjugating enzyme UBC13 (E2) and promotes methyl methanesulfonate (MMS)–induced PCNA polyubiquitination. The reduction of SHPRH by stable short hairpin RNA increases sensitivity to MMS and enhances genomic instability. Therefore, the yRad5/SHPRH-dependent pathway is a conserved and fundamental DNA repair mechanism that protects the genome from genotoxic stress

Xenopus: An alternative model system for identifying muco-active agents

The airway epithelium in human plays a central role as the first line of defense against environmental contaminants. Most respiratory diseases such as chronic obstructive pulmonary disease (COPD), asthma, and respiratory infections, disturb normal muco-ciliary functions by stimulating the hypersecretion of mucus. Several muco-active agents have been used to treat hypersecretion symptoms in patients. Current muco-active reagents control mucus secretion by modulating either airway inflammation, cholinergic parasympathetic nerve activities or by reducing the viscosity by cleaving crosslinking in mucin and digesting DNAs in mucus. However, none of the current medication regulates mucus secretion by directly targeting airway goblet cells. The major hurdle for screening potential muco-active agents that directly affect the goblet cells, is the unavailability of in vivo model systems suitable for high-throughput screening. In this study, we developed a high-throughput in vivo model system for identifying muco-active reagents using Xenopus laevis embryos. We tested mucus secretion under various conditions and developed a screening strategy to identify potential muco-regulators. Using this novel screening technique, we identified narasin as a potential muco-regulator. Narasin treatment of developing Xenopus embryos significantly reduced mucus secretion. Furthermore, the human lung epithelial cell line, Calu-3, responded similarly to narasin treatment, validating our technique for discovering muco-active reagent

Microhomology-mediated end joining induces hypermutagenesis at breakpoint junctions

Microhomology (MH) flanking a DNA double-strand break (DSB) drives chromosomal rearrangements but its role in mutagenesis has not yet been analyzed. Here we determined the mutation frequency of a URA3 reporter gene placed at multiple locations distal to a DSB, which is flanked by different sizes (15-, 18-, or 203-bp) of direct repeat sequences for efficient repair in budding yeast. Induction of a DSB accumulates mutations in the reporter gene situated up to 14-kb distal to the 15-bp MH, but more modestly to those carrying 18- and 203-bp or no homology. Increased mutagenesis in MH-mediated end joining (MMEJ) appears coupled to its slower repair kinetics and the extensive resection occurring at flanking DNA. Chromosomal translocations via MMEJ also elevate mutagenesis of the flanking DNA sequences 7.1 kb distal to the breakpoint junction as compared to those without MH. The results suggest that MMEJ could destabilize genomes by triggering structural alterations and increasing mutation burden

Tissue-specific DNA damage response in Mouse Whole-body irradiation

Background Genomic instability is a hallmark of various cancers, and DNA repair is an essential process for maintaining genomic integrity. Mammalian cells have developed various DNA repair mechanisms in response to DNA damage. Compared to the cellular response to DNA damage, the in vivo DNA damage response (DDR) of specific tissues has not been studied extensively. Objective In this study, mice were exposed to whole-body gamma (gamma)-irradiation to evaluate the specific DDR of various tissues. We treated male C57BL6/J mice with gamma-irradiation at different doses, and the DDR protein levels in different tissues were analyzed. Results The level of gamma-H2A histone family member X (gamma H2AX) increased in most organs after exposure to gamma-irradiation. In particular, the liver, lung, and kidney tissues showed higher gamma H2AX induction upon DNA damage, compared to that in the brain, muscle, and testis tissues. RAD51 was highly expressed in the testis, irrespective of irradiation. The levels of proliferating cell nuclear antigen (PCNA) and ubiquitinated PCNA increased in lung tissues upon irradiation, suggesting that the post-replication repair may mainly operate in the lungs in response to gamma-irradiation. Conclusion These results suggest that each tissue has a preferable repair mechanism in response to gamma-irradiation. Therefore, the understanding and application of tissue-specific DNA damage responses could improve the clinical approach of radiotherapy for treating specific cancers

PCNA Ubiquitination Is Important, But Not Essential for Translesion DNA Synthesis in Mammalian Cells

Translesion DNA synthesis (TLS) is a DNA damage tolerance mechanism in which specialized low-fidelity DNA polymerases bypass replication-blocking lesions, and it is usually associated with mutagenesis. In Saccharomyces cerevisiae a key event in TLS is the monoubiquitination of PCNA, which enables recruitment of the specialized polymerases to the damaged site through their ubiquitin-binding domain. In mammals, however, there is a debate on the requirement for ubiquitinated PCNA (PCNA-Ub) in TLS. We show that UV-induced Rpa foci, indicative of single-stranded DNA (ssDNA) regions caused by UV, accumulate faster and disappear more slowly in Pcna(K164R/K164R) cells, which are resistant to PCNA ubiquitination, compared to Pcna(+/+) cells, consistent with a TLS defect. Direct analysis of TLS in these cells, using gapped plasmids with site-specific lesions, showed that TLS is strongly reduced across UV lesions and the cisplatin-induced intrastrand GG crosslink. A similar effect was obtained in cells lacking Rad18, the E3 ubiquitin ligase which monoubiquitinates PCNA. Consistently, cells lacking Usp1, the enzyme that de-ubiquitinates PCNA exhibited increased TLS across a UV lesion and the cisplatin adduct. In contrast, cells lacking the Rad5-homologs Shprh and Hltf, which polyubiquitinate PCNA, exhibited normal TLS. Knocking down the expression of the TLS genes Rev3L, PolH, or Rev1 in Pcna(K164R/K164R) mouse embryo fibroblasts caused each an increased sensitivity to UV radiation, indicating the existence of TLS pathways that are independent of PCNA-Ub. Taken together these results indicate that PCNA-Ub is required for maximal TLS. However, TLS polymerases can be recruited to damaged DNA also in the absence of PCNA-Ub, and perform TLS, albeit at a significantly lower efficiency and altered mutagenic specificity

Mph1p promotes gross chromosomal rearrangement through partial inhibition of homologous recombination

Gross chromosomal rearrangement (GCR) is a type of genomic instability associated with many cancers. In yeast, multiple pathways cooperate to suppress GCR. In a screen for genes that promote GCR, we identified MPH1, which encodes a 3′–5′ DNA helicase. Overexpression of Mph1p in yeast results in decreased efficiency of homologous recombination (HR) as well as delayed Rad51p recruitment to double-strand breaks (DSBs), which suggests that Mph1p promotes GCR by partially suppressing HR. A function for Mph1p in suppression of HR is further supported by the observation that deletion of both mph1 and srs2 synergistically sensitize cells to methyl methanesulfonate-induced DNA damage. The GCR-promoting activity of Mph1p appears to depend on its interaction with replication protein A (RPA). Consistent with this observation, excess Mph1p stabilizes RPA at DSBs. Furthermore, spontaneous RPA foci at DSBs are destabilized by the mph1Δ mutation. Therefore, Mph1p promotes GCR formation by partially suppressing HR, likely through its interaction with RPA

Thrap3 promotes R-loop resolution via interaction with methylated DDX5

Transcription-replication conflicts lead to DNA damage and genomic instability, which are closely related to human diseases. A major source of these conflicts is the formation of R-loops, which consist of an RNA-DNA hybrid and a displaced single-stranded DNA. Although these structures have been studied, many aspects of R-loop biology and R-loop-mediated genome instability remain unclear. Here, we demonstrate that thyroid hormone receptor-associated protein 3 (Thrap3) plays a critical role in regulating R-loop resolution. In cancer cells, Thrap3 interacts with DEAD-box helicase 5 (DDX5) and localizes to R-loops. Arginine-mediated methylation of DDX5 is required for its interaction with Thrap3, and the Thrap3-DDX5 axis induces the recruitment of 5'-3' exoribonuclease 2 (XRN2) into R-loops. Loss of Thrap3 increases R-loop accumulation and DNA damage. These findings suggest that Thrap3 mediates resistance to cell death by preventing R-loop accumulation in cancer cells. Cancer: DNA damage associated with nucleic acid loops A nuclear protein appears to inhibit cancer cell death by preventing the accumulation of nucleic acid structures called R-loops. R-loops are by-products of transcription, comprising two misaligned DNA strands and one RNA strand. They are involved in gene expression, but also threaten genome integrity and have been linked to the onset of neurodegeneration and cancers. A team led by Jang Hyun Choi and Hyug Moo Kwon, Ulsan National Institute of Science and Technology, South Korea, explored the role of Thrap3, a nuclear protein involved in RNA splicing, in R-loop-associated DNA damage. They found that Thrap3 binds to an enzyme essential for resolving R-loops. When the team suppressed Thrap3 expression, they saw an increase in R-loops in both normal and cancer cells. This R-loop accumulation significantly inhibited the growth of breast cancer cells

Ring finger protein 126 (RNF126) suppresses ionizing radiation-induced p53-binding protein 1 (53BP1) focus formation

Cells have evolved sophisticated mechanisms to maintain genomic integrity in response to DNA damage. Ionizing radiation (IR)-induced DNA damage results in the formation of IR-induced foci (iRIF) in the nucleus. The iRIF formation is part of the DNA damage response (DDR), which is an essential signaling cascade that must be strictly regulated because either the loss of or an augmented DDR leads to loss of genome integrity. Accordingly, negative regulation of the DDR is as critical as its activation. In this study, we have identified ring finger protein 126 (RNF126) as a negative regulator of the DDR from a screen of iRIF containing 53BP1. RNF126 overexpression abolishes not only the formation of 53BP1 iRIF but also of RNF168, FK2, RAP80, and BRCA1. However, the iRIF formation of H2AX, MDC1, and RNF8 is maintained, indicating that RNF126 acts between RNF8 and RNF168 during the DDR. In addition, RNF126 overexpression consistently results in the loss of RNF168-mediated H2A monoubiquitination at lysine 13/15 and inhibition of the non-homologous end joining capability. Taken together, our findings reveal that RNF126 is a novel factor involved in the negative regulation of DDR, which is important for sustaining genomic integrity

Exploring the Role of a Novel Peptide from Allomyrina dichotoma Larvae in Ameliorating Lipid Metabolism in Obesity

The aim of this study was to identify an anti-obesity peptide from Allomyrina dichotoma and investigate the lipid metabolic mechanism. Enzymatically hydrolyzed A. dichotoma larvae were further separated using tangential flow filtration and consecutive chromatographic processes. Finally, an anti-obesity peptide that showed the highest inhibitory effect on lipid accumulation was obtained, and the sequence was Glu-Ile-Ala-Gln-Asp-Phe-Lys-Thr-Asp-Leu (EIA10). EIA10 decreased lipid aggregation in vitro and significantly reduced the accumulation of body weight gain, liver weight, and adipose tissue weight in high-fat-fed mice. Compared with the control group, the levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL), insulin, and homeostasis model assessment of insulin resistance (HOMA-IR) in the high-fat diet (HFD) group increased significantly, and the content of high-density lipoprotein cholesterol (HDL) in the serum decreased significantly. On the contrary, the levels of TC, TG, and insulin in the EIA10 group decreased significantly, and the HDL content increased significantly compared with the HFD group. Additionally, EIA10 dramatically decreased mRNA and protein levels of transcription factors involved in lipid adipogenesis. Taken together, our results suggest that EIA10 could be a promising agent for the treatment and prevention of obesity

Loss of adipose TET proteins enhances ??-adrenergic responses and protects against obesity by epigenetic regulation of ??3-AR expression

??-adrenergic receptor (??-AR) signaling plays predominant roles in modulating energy expenditure by triggering lipolysis and thermogenesis in adipose tissue, thereby conferring obesity resistance. Obesity is associated with diminished ??3-adrenergic receptor (??3-AR) expression and decreased ??-adrenergic responses, but the molecular mechanism coupling nutrient overload to catecholamine resistance remains poorly defined. Ten-eleven translocation (TET) proteins are dioxygenases that alter the methylation status of DNA by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine and further oxidized derivatives. Here, we show that TET proteins are pivotal epigenetic suppressors of ??3-AR expression in adipocytes, thereby attenuating the responsiveness to ??-adrenergic stimulation. Deletion of all three Tet genes in adipocytes led to increased ??3-AR expression and thereby enhanced the downstream ??-adrenergic responses, including lipolysis, thermogenic gene induction, oxidative metabolism, and fat browning in vitro and in vivo. In mouse adipose tissues, Tet expression was elevated after mice ate a high-fat diet. Mice with adipose-specific ablation of all TET proteins maintained higher levels of ??3-AR in both white and brown adipose tissues and remained sensitive to ??-AR stimuli under high-fat diet challenge, leading to augmented energy expenditure and decreased fat accumulation. Consequently, they exhibited improved cold tolerance and were substantially protected from diet-induced obesity, inflammation, and metabolic complications, including insulin resistance and hyperlipidemia. Mechanistically, TET proteins directly repressed ??3-AR transcription, mainly in an enzymatic activity-independent manner, and involved the recruitment of histone deacetylases to increase deacetylation of its promoter. Thus, the TET-histone deacetylase-??3-AR axis could be targeted to treat obesity and related metabolic diseases