NOTCH localizes to mitochondria through the TBC1D15-FIS1 interaction and is stabilized via blockade of E3 ligase and CDK8 recruitment to reprogram tumor-initiating cells.

The P53-destabilizing TBC1D15-NOTCH protein interaction promotes self-renewal of tumor-initiating stem-like cells (TICs); however, the mechanisms governing the regulation of this pathway have not been fully elucidated. Here, we show that TBC1D15 stabilizes NOTCH and c-JUN through blockade of E3 ligase and CDK8 recruitment to phosphodegron sequences. Chromatin immunoprecipitation (ChIP-seq) analysis was performed to determine whether TBC1D15-dependent NOTCH1 binding occurs in TICs or non-TICs. The TIC population was isolated to evaluate TBC1D15-dependent NOTCH1 stabilization mechanisms. The tumor incidence in hepatocyte-specific triple knockout (Alb::CreERT2;Tbc1d15Flox/Flox;Notch1Flox/Flox;Notch2Flox/Flox;HCV-NS5A) Transgenic (Tg) mice and wild-type mice was compared after being fed an alcohol-containing Western diet (WD) for 12 months. The NOTCH1-TBC1D15-FIS1 interaction resulted in recruitment of mitochondria to the perinuclear region. TBC1D15 bound to full-length NUMB and to NUMB isoform 5, which lacks three Ser phosphorylation sites, and relocalized NUMB5 to mitochondria. TBC1D15 binding to NOTCH1 blocked CDK8- and CDK19-mediated phosphorylation of the NOTCH1 PEST phosphodegron to block FBW7 recruitment to Thr-2512 of NOTCH1. ChIP-seq analysis revealed that TBC1D15 and NOTCH1 regulated the expression of genes involved in mitochondrial metabolism-related pathways required for the maintenance of TICs. TBC1D15 inhibited CDK8-mediated phosphorylation to stabilize NOTCH1 and protect it from degradation The NUMB-binding oncoprotein TBC1D15 rescued NOTCH1 from NUMB-mediated ubiquitin-dependent degradation and recruited NOTCH1 to the mitochondrial outer membrane for the generation and expansion of liver TICs. A NOTCH-TBC1D15 inhibitor was found to inhibit NOTCH-dependent pathways and exhibited potent therapeutic effects in PDX mouse models. This unique targeting of the NOTCH-TBC1D15 interaction not only normalized the perinuclear localization of mitochondria but also promoted potent cytotoxic effects against TICs to eradicate patient-derived xenografts through NOTCH-dependent pathways.

Reliable quantitative detection of uric acid in urine by surface-enhanced Raman spectroscopy with endogenous internal standard.

Abnormal levels of uric acid (UA) in urine serve as warning signs for gout and metabolic cardiovascular diseases, necessitating the monitoring of UA levels for early prevention. However, the current analytical methods employed suffer from limitations in terms of inadequate suitability for home-based applications and the requirement of non-invasive procedures. In this approach, creatinine, a metabolite with a constant excretion rate, was incorporated as an endogenous internal standard (e-IS) for calibration, presenting a rapid, pretreatment-free, and accurate strategy for quantitative determination of UA concentrations. By utilizing urine creatinine as an internal reference value to calibrate the signal fluctuation of surface-enhanced Raman spectroscopy (SERS) of UA, the quantitative accuracy can be significantly improved without the need for an external internal standard. Due to the influence of the medium, UA, which carries a negative charge, is selectively adsorbed by Au@Ag nanoparticles functionalized with hexadecyltrimethylammonium chloride (CTAC) in this system. Furthermore, a highly convenient detection method was developed, which eliminates the need for pre-processing and minimizes matrix interference by simple dilution. The method was applied to the urine detection of different volunteers, and the results were highly consistent with those obtained using the UA colorimetric kit (UACK). The detection time of SERS was only 30 s, which is 50 times faster than UACK. This quantitative strategy of using urinary creatinine as an internal standard to correct the SERS intensity of uric acid is also expected to be extended to the quantitative detection needs of other biomarkers in urine.

Adaptive spline surface fitting with arbitrary topological control mesh.

Reconstructing a spline surface from a given arbitrary topological triangle mesh is a fundamental and challenging problem in computer-aided design and engineering. This paper introduces a novel surface fitting method utilizing G-NURBS capable of handling control meshes with arbitrary topologies. This method employs adaptive control point adjustment, guided by the geometric attributes of the input model, ensuring precise representation of sharp features such as edges and corners. Two primary strategies are employed: a parameter correspondence approach designed for sharp features and a control mesh iterative refinement technique that incorporates geometrical feature information. The proposed method has been tested and evaluated on various CAD models to demonstrate its effectiveness. This method can achieve higher fitting accuracy while faithfully preserving the geometrical features with fewer control points.

Demographic dynamics and molecular evolution of the rare and endangered subsect. Gerardianae of Pinus: insights from chloroplast genomes and mitochondrial DNA markers.

MAIN CONCLUSION: The divergence of subsect. Gerardianae was likely triggered by the uplift of the Qinghai-Tibetan Plateau and adjacent mountains. Pinus bungeana might have probably experienced expansion since Last Interglacial period. Historical geological and climatic oscillations have profoundly affected patterns of nucleotide variability, evolutionary history, and species divergence in numerous plants of the Northern Hemisphere. However, how long-lived conifers responded to geological and climatic fluctuations in East Asia remain poorly understood. Here, based on paternally inherited chloroplast genomes and maternally inherited mitochondrial DNA markers, we investigated the population demographic history and molecular evolution of subsect. Gerardianae (only including three species, Pinus bungeana, P. gerardiana, and P. squamata) of Pinus. A low level of nucleotide diversity was found in P. bungeana (π was 0.00016 in chloroplast DNA sequences, and 0.00304 in mitochondrial DNAs). The haplotype-based phylogenetic topology and unimodal distributions of demographic analysis suggested that P. bungeana probably originated in the southern Qinling Mountains and experienced rapid population expansion since Last Interglacial period. Phylogenetic analysis revealed that P. gerardiana and P. squamata had closer genetic relationship. The species divergence of subsect. Gerardianae occurred about 27.18 million years ago (Mya) during the middle to late Oligocene, which was significantly associated with the uplift of the Qinghai-Tibetan Plateau and adjacent mountains from the Eocene to the mid-Pliocene. The molecular evolutionary analysis showed that two chloroplast genes (psaI and ycf1) were under positive selection, the genetic lineages of P. bungeana exhibited higher transition and nonsynonymous mutations, which were involved with the strongly environmental adaptation. These findings shed light on the population evolutionary history of white pine species and provide striking insights for comprehension of their species divergence and molecular evolution.

Resolving a nearly 90-year-old enigma: The rare Fagus chienii is conspecific with F. hayatae based on molecular and morphological evidence.

Taxonomic uncertainties of rare species often hinder effective prioritization for conservation. One such taxonomic uncertainty is the 90-year-old enigma of Fagus chienii. F. chienii was previously only known from the type specimens collected in 1935 in Pingwu County of Sichuan Province, China, and has long been thought to be on the verge of extinction. However, morphological similarities to closely related Fagus species have led many to question the taxonomic status of F. chienii. To clarify this taxonomic uncertainty, we used the newly collected samples to reconstruct a molecular phylogeny of Chinese Fagus species against the phylogenetic backbone of the whole genus using seven nuclear genes. In addition, we examined nine morphological characters to determine whether F. chienii is morphologically distinct from its putatively closest relatives (F. hayatae, F.longipetiolata, and F.lucida). Both morphological and phylogenetic analyses indicated that F. chienii is conspecific with F. hayatae. We recommended that F. chienii should not be treated as a separate species in conservation management. However, conservation strategies such as in situ protection and ex situ germplasm preservation should be adopted to prevent the peculiar "F. chienii" population from extinction.

Brumimicrobium oceani sp. nov., isolated from coastal sediment saline lake and environmental adaptability analysis.

A novel Gram-stain-negative, aerobic, non-motile, rod-shaped and orange-colored bacterium, designated as strain C305T, was isolated from marine sediment of the coast area of Weihai, China. Strain C305T growth occurs at 4-40 °C (optimally at 30-33 °C), pH 6.0-9.0 (optimally at pH 8.0) and with 0.5-10.0% (w/v) NaCl (optimum 1.5-3.0%). No growth is observed without NaCl. The major cellular fatty acids of strain C305T were identified as iso-C15:0, iso-C15:1G and iso-C17:0 3-OH. The major respiratory quinone was found to be MK-6, and the DNA G + C content was determined to be 35.5 mol%. The predominant polar lipids were mainly phosphatidylethanolamines (PE), unidentified aminophospholipids (APL), andunidentified lipid (L2). Phylogenetic analysis based on 16S rRNA gene sequences revealed that C305T was a member of the genus Brumimicrobium and had a 16S rRNA gene sequence similarity values of 96.9-98.0% with recognized Brumimicrobium species. On the basis of the phylogenetic and phenotypic evidences, strain C305T represents a novel species of the genus Brumimicrobium, for which the name Brumimicrobium oceani sp. nov. is proposed. The type strain is C305T (= KCTC 62371 T = MCCC 1H00297T).

MSI2 promotes translation of multiple IRES-containing oncogenes and virus to induce self-renewal of tumor initiating stem-like cells.

RNA-binding protein Musashi 2 (MSI2) is elevated in several cancers and is linked to poor prognosis. Here, we tested if MSI2 promotes MYC and viral mRNA translation to induce self-renewal via an internal ribosome entry sequence (IRES). We performed RIP-seq using anti-MSI2 antibody in tumor-initiating stem-like cells (TICs). MSI2 binds the internal ribosome entry site (IRES)-containing oncogene mRNAs including MYC, JUN and VEGFA as well as HCV IRES to increase their synthesis and promote self-renewal and tumor-initiation at the post-transcriptional level. MSI2 binds a lncRNA to interfere with processing of a miRNA that reduced MYC translation in basal conditions. Deregulation of this integrated MSI2-lncRNA-MYC regulatory loop drives self-renewal and tumorigenesis through increased IRES-dependent translation of MYC mRNA. Overexpression of MSI2 in TICs promoted their self-renewal and tumor-initiation properties. Inhibition of MSI2-RNA binding reduced HCV IRES activity, viral replication and liver hyperplasia in humanized mice predisposed by virus infection and alcohol high-cholesterol high-fat diet. Together MSI2, integrating the MYC oncogenic pathway, can be employed as a therapeutic target in the treatment of HCC patients. A hypothetical model shows that MSI2 binds and activates cap-independent translation of MYC, c-JUN mRNA and HCV through MSI2-binding to Internal Ribosome Entry Sites (IRES) resulting in upregulated MYC, c-JUN and viral protein synthesis and subsequent liver oncogenesis. Inhibitor of the interaction between MYC IRES and MSI2 reduces liver hyperplasia, viral mRNA translation and tumor formation.

Genotoxic effects of the major alkylation damage N7-methylguanine and methyl formamidopyrimidine.

Various alkylating agents are known to preferentially modify guanine in DNA, resulting in the formation of N7-alkylguanine (N7-alkylG) and the imidazole ring opened alkyl-formamidopyrimidine (alkyl-FapyG) lesions. Evaluating the mutagenic effects of N7-alkylG has been challenging due to the instability of the positively charged N7-alkylG. To address this issue, we developed a 2'-fluorine-mediated transition-state destabilization approach, which stabilizes N7-alkylG and prevents spontaneous depurination. We also developed a postsynthetic conversion of 2'-F-N7-alkylG DNA into 2'-F-alkyl-FapyG DNA. Using these methods, we incorporated site-specific N7-methylG and methyl-FapyG into pSP189 plasmid and determined their mutagenic properties in bacterial cells using the supF-based colony screening assay. The mutation frequency of N7-methylG was found to be less than 0.5%. Our crystal structure analysis revealed that N7-methylation did not significantly alter base pairing properties, as evidenced by a correct base pairing between 2'-F-N7-methylG and dCTP in Dpo4 polymerase catalytic site. In contrast, the mutation frequency of methyl-FapyG was 6.3%, highlighting the mutagenic nature of this secondary lesion. Interestingly, all mutations arising from methyl-FapyG in the 5'-GGT(methyl-FapyG)G-3' context were single nucleotide deletions at the 5'-G of the lesion. Overall, our results demonstrate that 2'-fluorination technology is a useful tool for studying the chemically labile N7-alkylG and alkyl-FapyG lesions.

The hybridization origin of the Chinese endemic herb genus Notopterygium (Apiaceae): Evidence from population genomics and ecological niche analysis.

Hybridization is recognized as a major force in species evolution and biodiversity formation, generally leading to the origin and differentiation of new species. Multiple hybridization events cannot easily be reconstructed, yet they offer the potential to study a number of evolutionary processes. Here, we used nuclear expressed sequence tag-simple sequence repeat and large-scale single nucleotide polymorphism variation data, combined with niche analysis, to investigate the putative independent hybridization events in Notopterygium, a group of perennial herb plants endemic to China. Population genomic analysis indicated that the four studied species are genetically well-delimited and that N. forrestii and N. oviforme have originated by hybridization. According to Approximate Bayesian Computation, the best-fit model involved the formation of N. forrestii from the crossing of N. franchetii and N. incisum, with N. forrestii further backcrossing to N. franchetii to form N. oviforme. The niche analyses indicated that niche divergence [likely triggered by the regional climate changes, particularly the intensification of East Asian winter monsoon, and tectonic movements (affecting both Qinghai-Tibetan Plateau and Qinling Mountains)] may have promoted and maintained the reproductive isolation among hybrid species. N. forrestii shows ecological specialization with respect to their parental species, whereas N. oviforme has completely shifted its niche. These results suggested that the climate and environmental factors together triggered the two-step hybridization of the East Asia herb plants. Our study also emphasizes the power of genome-wide SNPs for investigating suspected cases of hybridization, particularly unravelling old hybridization events.

Ultrasensitive detection of SARS-CoV-2 S protein with aptamers biosensor based on surface-enhanced Raman scattering.

A rapid and accurate diagnostic modality is essential to prevent the spread of SARS-CoV-2. In this study, we proposed a SARS-CoV-2 detection sensor based on surface-enhanced Raman scattering (SERS) to achieve rapid and ultrasensitive detection. The sensor utilized spike protein deoxyribonucleic acid aptamers with strong affinity as the recognition entity to achieve high specificity. The spherical cocktail aptamers-gold nanoparticles (SCAP) SERS substrate was used as the base and Au nanoparticles modified with the Raman reporter molecule that resonates with the excitation light and spike protein aptamers were used as the SERS nanoprobe. The SCAP substrate and SERS nanoprobes were used to target and capture the SARS-CoV-2 S protein to form a sandwich structure on the Au film substrate, which can generate ultra-strong "hot spots" to achieve ultrasensitive detection. Analysis of SARS-CoV-2 S protein was performed by monitoring changes in SERS peak intensity on a SCAP SERS substrate-based detection platform. This assay detects S protein with a LOD of less than 0.7 fg mL-1 and pseudovirus as low as 0.8 TU mL-1 in about 12 min. The results of the simulated oropharyngeal swab system in this study indicated the possibility of it being used for clinical detection, providing a potential option for rapid and accurate diagnosis and more effective control of SARS-CoV-2 transmission.

Impacts of the components of conservation agriculture on soil organic carbon and total nitrogen storage: A global meta-analysis.

Conservation agriculture (CA) can be an important strategy for improving soil organic carbon (SOC) and total nitrogen (TN). Numerous studies have examined SOC and TN dynamics in different cropping systems. However, there is some uncertainty regarding the relative impacts of some CA practices, and it is not always clear how other agricultural management, particularly nitrogen addition, interacts with these practices to influence SOC and TN sequestration. Thus, we conducted a global meta-analysis of 752 comparisons from 97 papers to analyze the impacts of nitrogen fertilizers and CA practices (namely crop diversification, minimal soil disturbance (no-tillage) and permanent soil cover), on SOC and TN content worldwide. Overall, our study showed the most significant increase of SOC [21.39 % (CI = 15.16 to 28.64)] and TN [54.34 % (CI = 26.19 to 96.69)] stock with CA practices compared to conventional practices in the 0-15 cm soil depth. It also showed a significant increase in SOC and TN stock with all the individual components of CA compared to conventional practices in the 0-15 cm soil depth. However, the impact of CA on SOC and TN is reduced in 0-60 cm depths compared to surface soil depths due to the limited input of crop residue deeper in the soil profile. Manure and manure mixed with mineral-N led to greater SOC sequestration [20.67 % (CI = 15.23 to 27.10) and 41.67 % (CI = 31.03 to 52.79), respectively] than mineral-N alone [9.08 % (CI = 6.44 to 11.83)]. Cropping systems that included legume residue decreased the C/N ratio. This highlights that adequate mineral-N fertilizer addition may also be required in conjunction with residue retention practices to improve SOC and TN content. Overall, these results show that CA systems that include legume residue and manure mixed with mineral-N have great potential to increase SOC and TN, particularly at 0-15 cm and 0-30 cm soil depth.

Novel Few-Shot Learning Neural Network for Predicting Carbohydrate-Active Enzyme Affinity Toward Fructo-Oligosaccharides.

The enzymatic activity of the microbiome toward carbohydrates in the human digestive system is of enormous health significance. Predicting how carbohydrates through food intake may affect the distribution and balance of gut microbiota remains a major challenge. Understanding the enzyme/substrate specificity relationship of the carbohydrate-active enzyme (CAZyme) encoded by the vast gut microbiome will be an important step to address this question. In this study, we seek to establish an in silico approach to studying the enzyme/substrate binding interaction. We focused on the key CAZyme and established a novel Poisson noise-based few-shot learning neural network (pFSLNN) for predicting the binding affinity of indigestible carbohydrates. This approach achieved higher accuracy than other classic FSLNNs, and we have also formulated new algorithms for feature generation using only a few amino acid (AA) sequences. Sliding bin regression is integrated with minimum redundancy maximum relevance for feature selection. The resulting pFSLNN is an efficient model to predict the binding affinity between CAZyme and common oligosaccharides. This model can be potentially applied to the binding affinity prediction of other protein/ligand interactions based on limited AA sequences.

Hypertonic Dextrose Stimulates Chondrogenic Cells to Deposit Collagen and Proliferate.

OBJECTIVE: Hypertonic dextrose (HD) injections (prolotherapy) for osteoarthritis are reported to reduce pain. Cartilage regeneration is hypothesized as a mechanism. This in vitro study identifies an HD concentration that stimulates chondrogenic cells to increase metabolic activity and assesses whether this concentration affects collagen deposition and proliferation. DESIGN: ATDC5 chondrogenic cells were cultured in normoglycemic DMEM/F12 medium, treated with concentrations of HD (4-400 mM), and assessed with PrestoBlue. Advanced light microscopy was used to conduct live imaging of collagen deposition through second harmonic generation microscopy (SHG) and proliferation via 2-photon excitation microscopy. Proliferation was additionally assessed with hemocytometer counts. RESULTS: A linear regression model found that, relative to the 4 mM baseline control, cells treated with 200 mM had a higher mean absorbance (P = 0.023) and cells treated with 250 mM were trending toward a higher mean absorbance (P = 0.076). Polynomial regression interpolated 240 mM as producing the highest average absorbance. Hemocytometer counts validated 250 mM as stimulating proliferation compared with the 4 mM control (P < 0.01). A concentration of 250 mM HD led to an increase in collagen deposition compared with that observed in control (P < 0.05). This HD concentration also led to increases in proliferation of ATDC5 cells relative to that of control (P < 0.001). CONCLUSIONS: A 250 mM HD solution appears to be associated with increased metabolic activity of chondrocytes, increased collagen deposition, and increased chondrocyte proliferation. These results support clinical prolotherapy research suggesting that intra-articular HD joint injections reduce knee pain. Further study of HD and cellular processes is warranted.

p53 destabilizing protein skews asymmetric division and enhances NOTCH activation to direct self-renewal of TICs.

Tumor-initiating stem-like cells (TICs) are defective in maintaining asymmetric cell division and responsible for tumor recurrence. Cell-fate-determinant molecule NUMB-interacting protein (TBC1D15) is overexpressed and contributes to p53 degradation in TICs. Here we identify TBC1D15-mediated oncogenic mechanisms and tested the tumorigenic roles of TBC1D15 in vivo. We examined hepatocellular carcinoma (HCC) development in alcohol Western diet-fed hepatitis C virus NS5A Tg mice with hepatocyte-specific TBC1D15 deficiency or expression of non-phosphorylatable NUMB mutations. Liver-specific TBC1D15 deficiency or non-p-NUMB expression reduced TIC numbers and HCC development. TBC1D15-NuMA1 association impaired asymmetric division machinery by hijacking NuMA from LGN binding, thereby favoring TIC self-renewal. TBC1D15-NOTCH1 interaction activated and stabilized NOTCH1 which upregulated transcription of NANOG essential for TIC expansion. TBC1D15 activated three novel oncogenic pathways to promote self-renewal, p53 loss, and Nanog transcription in TICs. Thus, this central regulator could serve as a potential therapeutic target for treatment of HCC.

Bypass of the Major Alkylative DNA Lesion by Human DNA Polymerase η.

A wide range of endogenous and exogenous alkylating agents attack DNA to generate various alkylation adducts. N7-methyl-2-deoxyguanosine (Fm7dG) is the most abundant alkylative DNA lesion. If not repaired, Fm7dG can undergo spontaneous depurination, imidazole ring-opening, or bypass by translesion synthesis DNA polymerases. Human DNA polymerase η (polη) efficiently catalyzes across Fm7dG in vitro, but its structural basis is unknown. Herein, we report a crystal structure of polη in complex with templating Fm7dG and an incoming nonhydrolyzable dCTP analog, where a 2'-fluorine-mediated transition destabilization approach was used to prevent the spontaneous depurination of Fm7dG. The structure showed that polη readily accommodated the Fm7dG:dCTP base pair with little conformational change of protein and DNA. In the catalytic site, Fm7dG and dCTP formed three hydrogen bonds with a Watson-Crick geometry, indicating that the major keto tautomer of Fm7dG is involved in base pairing. The polη-Fm7dG:dCTP structure was essentially identical to the corresponding undamaged structure, which explained the efficient bypass of the major methylated lesion. Overall, the first structure of translesion synthesis DNA polymerase bypassing Fm7dG suggests that in the catalytic site of Y-family DNA polymerases, small N7-alkylguanine adducts may be well tolerated and form the canonical Watson-Crick base pair with dCTP through their keto tautomers.

Promutagenicity of 8-Chloroguanine, A Major Inflammation-Induced Halogenated DNA Lesion.

Chronic inflammation is closely associated with cancer development. One possible mechanism for inflammation-induced carcinogenesis is DNA damage caused by reactive halogen species, such as hypochlorous acid, which is released by myeloperoxidase to kill pathogens. Hypochlorous acid can attack genomic DNA to produce 8-chloro-2'-deoxyguanosine (ClG) as a major lesion. It has been postulated that ClG promotes mutagenic replication using its syn conformer; yet, the structural basis for ClG-induced mutagenesis is unknown. We obtained crystal structures and kinetics data for nucleotide incorporation past a templating ClG using human DNA polymerase ß (polß) as a model enzyme for high-fidelity DNA polymerases. The structures showed that ClG formed base pairs with incoming dCTP and dGTP using its anti and syn conformers, respectively. Kinetic studies showed that polß incorporated dGTP only 15-fold less efficiently than dCTP, suggesting that replication across ClG is promutagenic. Two hydrogen bonds between syn-ClG and anti-dGTP and a water-mediated hydrogen bond appeared to facilitate mutagenic replication opposite the major halogenated guanine lesion. These results suggest that ClG in DNA promotes G to C transversion mutations by forming Hoogsteen base pairing between syn-ClG and anti-G during DNA synthesis.

Nanoparticle systems for cancer vaccine.

As effective tools for public health, vaccines prevent disease by priming the body's adaptive and innate immune responses against an infection. Due to advances in understanding cancers and their relationship with the immune system, there is a growing interest in priming host immune defenses for a targeted and complete antitumor response. Nanoparticle systems have shown to be promising tools for effective antigen delivery as vaccines and/or for potentiating immune response as adjuvants. Here, we highlight relevant physiological processes involved in vaccine delivery, review recent advances in the use of nanoparticle systems for vaccines and discuss pertinent challenges to viably translate nanoparticle-based vaccines and adjuvants for public use.

Structural and Kinetic Studies of the Effect of Guanine N7 Alkylation and Metal Cofactors on DNA Replication.

A wide variety of endogenous and exogenous alkylating agents attack DNA to preferentially generate N7-alkylguanine (N7-alkylG) adducts. Studies of the effect of N7-alkylG lesions on biological processes have been difficult in part because of complications arising from the chemical lability of the positively charged N7-alkylG, which can readily produce secondary lesions. To assess the effect of bulky N7-alkylG on DNA replication, we prepared chemically stable N7-benzylguanine (N7bnG)-containing DNA and evaluated nucleotide incorporation opposite the lesion by human DNA polymerase ß (polß), a model enzyme for high-fidelity DNA polymerases. Kinetic studies showed that the N7-benzyl-G lesion greatly inhibited dCTP incorporation by polß. The crystal structure of polß incorporating dCTP opposite N7bnG showed a Watson-Crick N7bnG:dCTP structure. The polß-N7bnG:dCTP structure showed an open protein conformation, a relatively disordered dCTP, and a lack of catalytic metal, which explained the inefficient nucleotide incorporation opposite N7bnG. This indicates that polß is sensitive to major groove adducts in the templating base side and deters nucleotide incorporation opposite bulky N7-alkylG adducts by adopting a catalytically incompetent conformation. Substituting Mg2+ for Mn2+ induced an open-to-closed conformational change due to the presence of catalytic metal and stably bound dCTP and increased the catalytic efficiency by ∼10-fold, highlighting the effect of binding of the incoming nucleotide and catalytic metal on protein conformation and nucleotidyl transfer reaction. Overall, these results suggest that, although bulky alkyl groups at guanine-N7 may not alter base pairing properties of guanine, the major groove-positioned lesions in the template could impede nucleotidyl transfer by some DNA polymerases.

Crystal Structure of Apo MEF2B Reveals New Insights in DNA Binding and Cofactor Interaction.

The myocyte enhancer factor 2 (MEF2) family of transcription factors plays important roles in developmental processes and adaptive responses. Although MEF2 proteins are known to bind DNA in the nucleus to regulate specific gene expression, there are reports that show that MEF2 also functions in the cytoplasm. Previous structural studies of MEF2 focused exclusively on DNA-bound MEF2 with and without various corepressors or coactivators. While these studies have established a comprehensive structural model of DNA recognition and cofactor recruitment by MEF2, the structure of MEF2 not bound to DNA, which include cytoplasmic MEF2 and free MEF2 in the nucleus, is unknown. Here we determined the structure of the MADS-box/MEF2 domain of MEF2B without DNA nor cofactor. The Apo structure of MEF2B reveals a largely preformed DNA binding interface that may be important for recognizing the shape of DNA from the minor groove side. In addition, our structure also reveals that the C-terminal helix of the MEF2-specific domain could flip up to bind to the hydrophobic groove that serves as the binding sites of MEF2 transcription cofactors. These observations shed new insights into DNA binding and cofactor interaction by MEF2 proteins.

The Cancer Mutation D83V Induces an α-Helix to ß-Strand Conformation Switch in MEF2B.

MEF2B is a major target of somatic mutations in non-Hodgkin lymphoma. Most of these mutations are non-synonymous substitutions of surface residues in the MADS-box/MEF2 domain. Among them, D83V is the most frequent mutation found in tumor cells. The link between this hotspot mutation and cancer is not well understood. Here we show that the D83V mutation induces a dramatic α-helix to ß-strand switch in the MEF2 domain. Located in an α-helix region rich in ß-branched residues, the D83V mutation not only removes the extensive helix stabilization interactions but also introduces an additional ß-branched residue that further shifts the conformation equilibrium from α-helix to ß-strand. Cross-database analyses of cancer mutations and chameleon sequences revealed a number of well-known cancer targets harboring ß-strand favoring mutations in chameleon α-helices, suggesting a commonality of such conformational switch in certain cancers and a new factor to consider when stratifying the rapidly expanding cancer mutation data.