Methylguanine Research Articles

DNA mismatch repair controls the mutagenicity of Polymerase ζ-dependent translesion synthesis at methylated guanines

By replicating damaged nucleotides, error-prone DNA translesion synthesis (TLS) enables the completion of replication, albeit at the expense of fidelity. TLS of helix-distorting DNA lesions, that usually have reduced capacity of basepairing, comprises insertion opposite the lesion followed by extension, the latter in particular by polymerase ζ (Pol ζ). However, little is known about involvement of Pol ζ in TLS of non- or poorly-distorting, but miscoding, lesions such as O6-methyldeoxyguanosine (O6-medG). Using purified Pol ζ we describe that the enzyme can misincorporate thymidine opposite O6-medG and efficiently extend from terminal mismatches, suggesting its involvement in the mutagenicity of O6-medG. Surprisingly, O6-medG lesions induced by the methylating agent N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) appeared more, rather than less, mutagenic in Pol ζ-deficient mouse embryonic fibroblasts (MEFs) than in wild type MEFs. This suggested that in vivo Pol ζ participates in non-mutagenic TLS of O6-medG. However, we found that the Pol ζ-dependent misinsertions at O6-medG lesions are efficiently corrected by DNA mismatch repair (MMR), which masks the error-proneness of Pol ζ. We also found that the MNNG-induced mutational signature is determined by the adduct spectrum, and modulated by MMR. The signature mimicked single base substitution signature 11 in the catalogue of somatic mutations in cancer, associated with treatment with the methylating drug temozolomide. Our results unravel the individual roles of the major contributors to methylating drug-induced mutagenesis. Moreover, these results warrant caution as to the classification of TLS as mutagenic or error-free based on in vitro data or on the analysis of mutations induced in MMR-proficient cells.

Regioselectivity and physical nature of the interactions between (methyl)guanine with HCl and CH3OH

A comprehensive study of the hydrogen bonding interactions between guanine (G) and methyl guanine derivatives (MGs) in the presence of HCl and MeOH is carried out at B3LYP, B3LYP-D3 and M062X/6–311 + + G(d.p) levels using molecular electrostatic potential, natural bond orbital, and symmetry adapted perturbation theory. Making use of these state-of-the-art techniques, this study attempts to elucidate the chemical bonding, regioselectivity, and physical nature of the interactions responsible for the stability of (M)G…L (L = HCl, MeOH) acid–base complexes. Our calculations reveal that 1-G, 3-MG, and 5-MG interact more strongly with MeOH than HCl due to the positive hydrogen bond cooperativity. Furthermore, the carbonyl site on G is found to be the most reactive site, while methyl substitution increases the basicity of the nucleobase, thus yieding more stable complexes. The strongest H-bond interaction in G-complexes is found when HCl and MeOH attack the carbonyl group in anti-position. Finally, energy decomposition analyses through the symmetry-adapted perturbation theory reveal that most complexes are mainly stabilized via electrostatic interactions. The energy difference between complex isomers shows a competition between 3-HCl-G (MG) and 4-HCl-G (MG) at ∆G level where thermal, BSSE and entropy terms are included.

Temozolomide-fatty acid conjugates for glioblastoma multiforme: In vitro and in vivo evaluation.

Glioblastoma multiforme (GBM) is the deadliest brain tumor with a poor prognosis and limited therapeutic options. Temozolomide (TMZ) is the first-line chemotherapeutic agent used for the treatment of GBM; however, it suffers from several limitations, including short half-life, rapid metabolism, <1% brain bioavailability, methyl guanine methyl transferase (MGMT) based chemoresistance, and hematological toxicities. Several approaches have been adopted to overcome these limitations, particularly by using nanotechnology-based systems, but its physicochemical properties make TMZ challenging to load into these nanocarriers. In the current research, we conjugated TMZ with different fatty acids, i.e., linoleic acid (LA), oleic acid (OA), and palmitic acid (PA), to obtain TMZ-fatty acid conjugates, which are comparatively hydrophobic, less prone to degradation and potent. These conjugates were thoroughly characterized using 1H NMR spectroscopy, high-resolution mass spectrometry (HR-MS), and reverse phase-high performance liquid chromatography (RP-HPLC). The synthesized conjugates, namely Temozolomide-oleic acid (TOA,6R1), Temozolomide-linoleic acid (TLA, 6R2), and Temozolomide-palmitic acid (TPA, 6R3), showed an IC50 of 101.4, 67.97, and 672.04μM, respectively in C6 cells and 428.257, 366.43 and 413.69μM, respectively in U87-MG cells. On the other hand, the free TMZ showed an IC50 of >1000μM and 564.23μM in C6 and U87-MG, respectively. Further, the in vivo efficacy of the TMZ-fatty acid conjugates was evaluated in the C6-induced orthotropic rat glioblastoma model, wherein the TMZ-fatty acid conjugate showed improved survival rate (1.6 folds) and overall health of the animals. Collectively, the conjugation of fatty acids with TMZ improves its anticancer potential against glioblastoma multiforme (GBM).

TROP2 translation mediated by dual m6A/m7G RNA modifications promotes bladder cancer development

RNA modifications, including adenine methylation (m6A) of mRNA and guanine methylation (m7G) of tRNA, are crucial for the biological function of RNA. However, the mechanism underlying the translation of specific genes synergistically mediated by dual m6A/m7G RNA modifications in bladder cancer (BCa) remains unclear. We demonstrated that m6A methyltransferase METTL3-mediated programmable m6A modification of oncogene trophoblast cell surface protein 2 (TROP2) mRNA promoted its translation during malignant transformation of bladder epithelial cells. m7G methyltransferase METTL1 enhanced TROP2 translation by mediating m7G modification of certain tRNAs. TROP2 protein inhibition decreased the proliferation and invasion of BCa cells in vitro and in vivo. Moreover, synergistical knockout of METTL3/METTL1 inhibited BCa cell proliferation, migration, and invasion; however, TROP2 overexpression partially abrogated its effect. Furthermore, TROP2 expression was significantly positively correlated with the expression levels of METTL3 and METTL1 in BCa patients. Overall, our results revealed that METTL3/METTL1-mediated dual m6A/m7G RNA modifications enhanced TROP2 translation and promoted BCa development, indicating a novel RNA epigenetic mechanism in BCa.

Cognitive functioning in a cohort of high-grade glioma patients.

High grade gliomas are associated with cognitive problems. The aim of the study was to investigate cognitive functioning in a cohort of patients with high grade glioma, according to isocitrate dehydrogenase (IDH) and methyl guanine methyl transferase (MGMT) status and other clinical characteristics. The patients with the high-grade glioma treated in Slovenia in given period of time were included in study. Postoperatively they completed neuropsychological assessment consisting of Slovenian Verbal Learning Test, Slovenian Controlled Oral Word Association Test, Trail Making Test Part A and B and self-evaluation questionnaire. We analysed results (z-scores and dichotomized results) also according to IDH mutation and MGMT methylation. We examined differences between groups using T-test, Mann-Whitney U, χ2 and Kendall's Tau tests. Out of 275 patients in the cohort, we included 90. Forty-six percent of patients were unable to participate due to poor performance status and other conditions related to tumour. Patients with the IDH mutation were younger, with better performance status, larger proportions of grade III tumours and MGMT methylation. In this group cognitive functioning is significantly better in the domains of immediate recall, short delayed recall and delayed recall, and in the fields of executive functioning and recognition. There were no differences in cognitive functioning in regard to MGMT status. Grade III tumours were associated with more frequent MGMT methylation. Self-assessment proved week tool, associated only with immediate recall. We found no differences in cognitive functioning according to MGMT status, but cognition was better when IDH mutation was present. In a cohort study of patients with high-grade glioma, almost half were unable to participate in a study, which points to an overrepresentation of patients with better cognitive functioning in the research.

Abstract 6209: O6-methylguanine as the junction of MGMT and PARP1-mediated repair pathways

Abstract Temozolomide is the first-choice DNA alkylating agent and has been commonly used in oncology over the last several decades. The cytotoxicity that temozolomide elicits through the methylation of guanine and adenine residues often becomes the limiting factor in effective treatment. Despite growing evidence of dysregulated alkylating DNA damage repair as a driving force of genome instability leading to cancer, neurological diseases, and premature aging, little is currently known about the coordinated role of PARP1 and MGMT (O6-methylguanine methyltransferase) enzymes in the repair of temozolomide-induced methylated DNA lesions. A major PARP1 function in DNA damage is facilitation of repair of the methylated DNA lesions, N6-methyladenine and N7-methylguanine, via base excision repair (BER); while MGMT restores guanine in O6-methylguanine (O6meG), the most cytotoxic adduct, by a one-step catalysis. It is generally thought that BER and MGMT represent two distinct mechanisms for removing DNA damage induced by alkylating agents; however, using a number of advanced cell-free and cell-based approaches, we provided evidence for direct (DNA-independent) and indirect (through PARylation) interaction between PARP1 and MGMT and demonstrated a functional crosstalk between these repair pathways. Particularly, O6meG repair activity is increased once PARP1 PARylates MGMT. Further, longer (more clinically relevant) exposure to temozolomide induced stronger MGMT PARylation in cells, indicating that the PARP1-MGMT interaction is important for enhanced O6meG repair and cell survival. As PARP1-MGMT complex forms in a variety of cancer cell types, our findings have strong implications for the development of an effective cancer therapy for cells dependent on PARP1 and MGMT mediated DNA repair. Citation Format: Jodie D. Cropper, Dauren S. Alimbetov, Kevin T. Brown, Andrew Robles, Rostislav I. Likhotvorik, James T. Guerra, Yidong Chen, Youngho Kwon, Raushan T. Kurmasheva. O6-methylguanine as the junction of MGMT and PARP1-mediated repair pathways. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6209.

Pseudo-Complementary G:C Base Pair for Mixed Sequence dsDNA Invasion and Its Applications in Diagnostics (SARS-CoV-2 Detection).

Pseudo-complementary oligonucleotides contain artificial nucleobases designed to reduce duplex formation in the pseudo-complementary pair without compromising duplex formation to targeted (complementary) oligomers. The development of a pseudo-complementary A:T base pair, Us:D, was important in achieving dsDNA invasion. Herein, we report pseudo-complementary analogues of the G:C base pair leveraged on steric and electrostatic repulsion between the cationic phenoxazine analogue of cytosine (G-clamp, C+) and N-7 methyl guanine (G+), which is also cationic. We show that while complementary peptide nucleic acids (PNA) form a much more stable homoduplex than the PNA:DNA heteroduplex, oligomers based on pseudo-C:G complementary PNA favor PNA:DNA hybridization. We show that this enables dsDNA invasion at physiological salt concentration and that stable invasion complexes are obtained with low equivalents of PNAs (2-4 equiv). We harnessed the high yield of dsDNA invasion for the detection of RT-RPA amplicon using a lateral flow assay (LFA) and showed that two strains of SARS-CoV-2 can be discriminated owing to single nucleotide resolution.

Important Requirements for Desorption/Ionization Mass Spectrometric Measurements of Temozolomide-Induced 2'-Deoxyguanosine Methylations in DNA.

In clinical pharmacology, drug quantification is mainly performed from the circulation for pharmacokinetic purposes. Finely monitoring the chemical effect of drugs at their chemical sites of action for pharmacodynamics would have a major impact in several contexts of personalized medicine. Monitoring appropriate drug exposure is particularly challenging for alkylating drugs such as temozolomide (TMZ) because there is no flow equilibrium that would allow reliable conclusions to be drawn about the alkylation of the target site from plasma concentrations. During the treatment of glioblastoma, it appears, therefore, promising to directly monitor the alkylating effect of TMZ rather than plasma exposure, ideally at the site of action. Mass spectrometry (MS) is a method of choice for the quantification of methylated guanines and, more specifically, of O6-methylguanines as a marker of TMZ exposure at the site of action. Depending on the chosen strategy to analyze modified purines and 2'-deoxynucleosides, the analysis of methylated guanines and 2'-deoxyguanosines is prone to important artefacts due to the overlap between masses of (i) guanines from DNA and RNA, and (ii) different methylated species of guanines. Therefore, the specific analysis of O6-methyl-2'deoxyguanosine, which is the product of the TMZ effect, is highly challenging. In this work, we report observations from matrix-assisted laser desorption/ionization (MALDI), and desorption electrospray ionization (DESI) MS analyses. These allow for the construction of a decision tree to initiate studies using desorption/ionization MS for the analysis of 2'-deoxyguanosine methylations induced by TMZ.

CRISPR/Cas9-guided knockout of eIF4E improves Wheat yellow mosaic virus resistance without yield penalty.

CRISPR/Cas9-guided knockout of eIF4E improves Wheat yellow mosaic virus resistance without yield penalty.

Multiparametric Magnetic Resonance Imaging Correlates of Isocitrate Dehydrogenase Mutation in WHO high-Grade Astrocytomas.

Isocitrate dehydrogenase (IDH) mutation and O-6 methyl guanine methyl transferase (MGMT) methylation are surrogate biomarkers of improved survival in gliomas. This study aims at studying the ability of semantic magnetic resonance imaging (MRI) features to predict the IDH mutation status confirmed by the gold standard molecular tests. The MRI of 148 patients were reviewed for various imaging parameters based on the Visually AcceSAble Rembrandt Images (VASARI) study. Their IDH status was determined using immunohistochemistry (IHC). Fisher's exact or chi-square tests for univariate and logistic regression for multivariate analysis were used. Parameters such as mild and patchy enhancement, minimal edema, necrosis &lt; 25%, presence of cysts, and less rCBV (relative cerebral blood volume) correlated with IDH mutation. The median age of IDH-mutant and IDH-wild patients were 34 years (IQR: 29-43) and 52 years (IQR: 45-59), respectively. Mild to moderate enhancement was observed in 15/19 IDH-mutant patients (79%), while 99/129 IDH-wildtype (77%) had severe enhancement (p-value &lt;0.001). The volume of edema with respect to tumor volume distinguished IDH-mutants from wild phenotypes (peritumoral edema volume &lt; tumor volume was associated with higher IDH-mutant phenotypes; p-value &lt; 0.025). IDH-mutant patients had a median rCBV value of 1.8 (IQR: 1.4-2.0), while for IDH-wild phenotypes, it was 2.6 (IQR: 1.9-3.5) {p-value = 0.001}. On multivariate analysis, a cut-off of 25% necrosis was able to differentiate IDH-mutant from IDH-wildtype (p-value &lt; 0.001), and a cut-off rCBV of 2.0 could differentiate IDH-mutant from IDH-wild phenotypes (p-value &lt; 0.007). Semantic imaging features could reliably predict the IDH mutation status in high-grade gliomas. Presurgical prediction of IDH mutation status could help the treating oncologist to tailor the adjuvant therapy or use novel IDH inhibitors.

Untargeted Metabolomics Profiling Reveals Beneficial Changes in Milk of Sows Supplemented with Fermented Compound Chinese Medicine Feed Additive.

Different untargeted metabolomics approaches were used to identify the differential metabolites between milk samples collected from two groups. Sows were supplemented with fermented compound Chinese medicine feed additive at levels of 0 g/d/sow (control group, n = 10) and 50 g/d/sow (experimental group, n = 10), respectively, from d 104 of gestation to d 25 of lactation, samples of colostrum and mature milk were collected. Data indicated that supplementing fermented compound Chinese medicine feed additive to sows significantly increased the concentrations of quercetin, pinocembrin, chlorogenic acid, methyl succinic acid, L-tryptophan, adenosine, guanine, arteannuin, ferulic acid, echimidine N-oxide, pogostone and kynurenine in the colostrum and inosine, guanosine, benzene-1,2,4-triol, hypoxanthine, adenine, trehalose 6-phosphate in mature milk, respectively. Seven pathways (flavone and flavanol biosynthesis, galactose metabolism, phenylpropanoid biosynthesis, stilbenoid and gingerol biosynthesis, flavonoid biosynthesis, ABC transporters and purine metabolism) in colostrum and two pathways (sucrose metabolism and retrograde endocannabinoid signaling) in mature milk were significantly enriched in the experimental group compared to control group, respectively. The supplementation of fermented compound Chinese medicine feed additive to sows increased the level of antibacterial and anti-inflammatory ingredients in milk and the findings of this study hint that supplementation with fermented compound Chinese medicine feed additive in sows is beneficial for the improvement of milk quality.

Thymoquinone induces apoptosis in temozolomide-resistant glioblastoma cells via the p38 mitogen-activated protein kinase signaling pathway.

Temozolomide (TMZ) can cross the blood-brain barrier (BBB) and deliver methyl groups to the purine (guanine) bases of DNA, leading to mispairing during DNA replication and subsequent cell death. However, increased expression of the repair enzyme methyl guanine methyltransferase (MGMT), which removes methyl groups from purine bases, counteracts methylation by TMZ. We evaluated the anticancer potential of thymoquinone (TQ), a hydrophobic flavonoid that inhibits resistance and induces apoptosis in various cancer cells, both in vitro and in vivo. In vitro experiments showed that compared with the Hs683 and M059J cell lines, U251 cells were more sensitive to TMZ. Compared to U251 cells, U251R cells, a TMZ drug-resistant strain established in this study, are characterized by increased expression of phosphorylated extracellular signal-regulated kinase (p-ERK) and MGMT. TQ treatments induced apoptosis in all cell lines. The p38 mitogen-activated protein kinase signal pathway was mainly activated in U251 and U251R cells; however, p-ERK and MGMT upregulation could not suppress TQ effects. Furthermore, si-p38 pretreatment of U251R cells in TQ treatments inhibited cell apoptosis. We speculate that TQ contributed to the phosphorylation and activation of p38, but not of ERK-induced apoptosis (irrespective of TMZ resistance). In vivo, U251R-derived tumors subcutaneously inoculated in nude mice exhibited significant tumor volume reduction after TQ or TQ + TMZ cotreatments. High-performance liquid chromatography assay confirmed the presence of TQ in murine brain tissues. Our findings demonstrate that TQ can effectively cross the BBB and function alone or in combination with TMZ to treat glioblastoma.

Contributing Factors for Mutagenic DNA Lesion Bypass by DNA Polymerase Eta (polη)

The integrity of DNA replication is under constant threat from various exogenous and endogenous factors along with some epigenetic factors. When there is damage to the genome, cells respond to the damage in two major ways, DNA damage repair and DNA damage tolerance. One of the major mechanisms for DNA damage tolerance is DNA lesion bypass, which is performed by specific DNA polymerases called Y-family DNA polymerases including DNA polymerase eta (polη). Ever since the discovery of polη’s unique role in bypassing cyclobutane pyrimidine dimer (CPD), a wide range of DNA lesions have been experimentally shown to be bypassed by polη. The structural study of polη was greatly boosted by the first elucidation of the N-terminal catalytic domain of polη by X-ray crystallography in 2010. Ever since, a lot of polη catalytic domain crystal structures have been published, which were complexed with an incoming nucleotide and a lesion containing DNA including pyrimidine dimers, cisplatin GpG adduct, 8-oxoguanine (oxoG), 8-oxoadenine (oxoA), N7-methylguanine (N7mG), O6-methylguanine (O6mG), hypoxanthine (HX), and many others. Though polη’s active site is known to be rigid with few conformational changes, there are several contributing factors that could facilitate the lesion bypass such as catalytic metals, syn–anti conformational equilibrium, tautomerization, and specific residues of polη. Each of these components are discussed in detail in this review.

Substitutional Sodium Doping on Tungsten Ditelluride: A First-principles Study

Methylation at O6 atom of guanine is a type of DNA damage which can cause a cancer. This damage at O6 atom of guanine in a DNA can be transferred to SG atom of cysteine in O6-alkylguanine-DNA alkyltransferase (AGT). Flipping out of methylated guanine from its base stack is essential to give off the methyl adduct (CH3) to AGT. AGT receives the methyl adduct at cysteine leaving guanine demethylated, but still in flipped out orientation. The repair mechanism of DNA would be completed only when the extrahelically flipped guanine returns back into the base stack, which is considered as the final step of the DNA repair mechanism. Here, the intrahelical flipping mechanism of repaired guanine has been studied. The work is further extended to examine the stability of hydrogen bonds between guanine and its pair partner cytosine. The overall result shows that intrahelical rotation of repaired guanine is possible and the base pairing is stable as the ordinary hydrogen bonding. BIBECHANA 19(2022)184-194

Flipping Back of Extrahelical Guanine after Methyl Repair

Methylation at O6 atom of guanine is a type of DNA damage which can cause a cancer. This damage at O6 atom of guanine in a DNA can be transferred to SG atom of cysteine in O6-alkylguanine-DNA alkyltransferase (AGT). Flipping out of methylated guanine from its base stack is essential to give off the methyl adduct (CH3) to AGT. AGT receives the methyl adduct at cysteine leaving guanine demethylated, but still in flipped out orientation. The repair mechanism of DNA would be completed only when the extrahelically flipped guanine returns back into the base stack, which is considered as the final step of the DNA repair mechanism. Here, the intrahelical flipping mechanism of repaired guanine has been studied. The work is further extended to examine the stability of hydrogen bonds between guanine and its pair partner cytosine. The overall result shows that intrahelical rotation of repaired guanine is possible and the base pairing is stable as the ordinary hydrogen bonding. BIBECHANA 19 (2022) 201-207

Abstract 4034: ALKBH3 deficiency and liability to alkylating agents in CRC models

Abstract Colorectal cancer (CRC) is the third most frequent malignancy and the second leading cause of cancer death worldwide. Despite the increase in therapeutic options for the treatment of metastatic CRC (mCRC), cytotoxic chemotherapy using fluoropyrimidines, oxaliplatin, and irinotecan still constitutes the mainstay of the treatment. These genotoxic drugs function by inducing direct or indirect DNA damage, which leads to cell cycle block and cell death. We previously showed that the presence of transcriptional repression of O-6-methylguanine-DNA methyltransferase (MGMT), a gene involved in direct repair of methylated guanines, confers increased sensitivity to temozolomide treatment -a DNA alkylating agent- in mCRC patients. We conducted a screening for down-regulation of DNA repair genes in a large panel of 245 molecularly annotated human colorectal cancer cell lines with the aim to identify other pharmacological liabilities to alkylating agents. 5 cell lines (2%) were identified to be significantly downregulated for alpha-ketoglutarate-dependent dioxygenase alkB homolog 3 (ALKBH3). ALKBH3 encodes a DNA dioxygenase that counteracts toxic alkylation damage inflicted by SN2 agents such as methyl methane sulfonate (MMS), and is particularly active at repairing 3-methyl-Cytosine (3meC) residues on single stranded DNA (ssDNA). Previous reports have shown that epigenetic loss of ALKBH3 has prognostic implication in lung cancer, Hodgkin lymphoma, Hepatocellular carcinoma (HCC) and breast cancer. However, no predictive value is currently known for ALKBH3 downregulation with respect to alkylating agents treatment. In order to evaluate the pharmacogenomics of ALKBH3-deficient CRC we firstly validated the absence of ALKBH3 protein by WB analysis in our CRC cell panel, then we confirmed ALKBH3 CpG promoter methylation, as previously described in literature for breast cancer models. Subsequently, we evaluated the sensitivity to several alkylating agents, including temozolomide, cisplatin, MMS, dacarbazine, lomustine, and carmustine in our panel of ALKBH3-null cell lines in comparison with a panel of ALKBH3-proficient cell lines. One MGMT-methylated CRC cell line was used as positive control for high sensitivity to temozolomide. We performed both short term (96 hours) and long term (up to 14 days) cell viability assay, using as readout for cell viability CellTitre Glow (CTG) and crystal violet staining, respectively. No statistically significant differences in sensitivity to alkylating agents was found in ALKBH3-null cell lines compared to ALKBH3-proficient cell lines, irrespectively from other genetic biomarkers such as microsatellite status or RAS/BRAF mutations. Taken together, these data demonstrate that ALKBH3 expression is not a candidate predictive biomarker for sensitivity to alkylating agents in CRC, most likely because of a high degree of redundancy of the systems involved in direct repair of alkylated DNA. Citation Format: Pietro Paolo Vitiello, Elisa Mariella, Pietro Andrei, Vito Amodio, Giovanni Germano, Annalisa Lorenzato, Carlotta Cancelliere, Alberto Bardelli. ALKBH3 deficiency and liability to alkylating agents in CRC models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4034.

Cell type-specific abnormalities of central nervous system in myotonic dystrophy type 1.

Myotonic dystrophy type 1 is a multisystem genetic disorder involving the muscle, heart and CNS. It is caused by toxic RNA transcription from expanded CTG repeats in the 3′-untranslated region of DMPK, leading to dysregulated splicing of various genes and multisystemic symptoms. Although aberrant splicing of several genes has been identified as the cause of some muscular symptoms, the pathogenesis of CNS symptoms prevalent in patients with myotonic dystrophy type 1 remains unelucidated, possibly due to a limitation in studying a diverse mixture of different cell types, including neuronal cells and glial cells. Previous studies revealed neuronal loss in the cortex, myelin loss in the white matter and the presence of axonal neuropathy in patients with myotonic dystrophy type 1. To elucidate the CNS pathogenesis, we investigated cell type-specific abnormalities in cortical neurons, white matter glial cells and spinal motor neurons via laser-capture microdissection. We observed that the CTG repeat instability and cytosine–phosphate–guanine (CpG) methylation status varied among the CNS cell lineages; cortical neurons had more unstable and longer repeats with higher CpG methylation than white matter glial cells, and spinal motor neurons had more stable repeats with lower methylation status. We also identified splicing abnormalities in each CNS cell lineage, such as DLGAP1 in white matter glial cells and CAMKK2 in spinal motor neurons. Furthermore, we demonstrated that aberrant splicing of CAMKK2 is associated with abnormal neurite morphology in myotonic dystrophy type 1 motor neurons. Our laser-capture microdissection-based study revealed cell type-dependent genetic, epigenetic and splicing abnormalities in myotonic dystrophy type 1 CNS, indicating the significant potential of cell type-specific analysis in elucidating the CNS pathogenesis.

Repurposing the Damage Repair Protein Methyl Guanine Methyl Transferase as a Ligand Inducible Fusion Degron.

We successfully repurpose the DNA repair protein methylguanine methyltransferase (MGMT) as an inducible degron for protein fusions. MGMT is a suicide protein that removes alkyl groups from the O6 position of guanine (O6G) and is thereafter quickly degraded by the ubiquitin proteasome pathway (UPP). Starting with MGMT pseudosubstrates (benzylguanine and lomeguatrib), we first demonstrate that these lead to potent MGMT depletion while affecting little else in the proteome. We then show that fusion proteins of MGMT undergo rapid UPP-dependent degradation in response to pseudosubstrates. Mechanistic studies confirm the involvement of the UPP, while revealing that at least two E3 ligase classes can degrade MGMT depending on cell-line and expression type (native or ectopic). We also demonstrate the technique's versatility with two clinically relevant examples: degradation of KRASG12C and a chimeric antigen receptor.

Versatile cell-based assay for measuring DNA alkylation damage and its repair

DNA alkylation damage induced by environmental carcinogens, chemotherapy drugs, or endogenous metabolites plays a central role in mutagenesis, carcinogenesis, and cancer therapy. Base excision repair (BER) is a conserved, front line DNA repair pathway that removes alkylation damage from DNA. The capacity of BER to repair DNA alkylation varies markedly between different cell types and tissues, which correlates with cancer risk and cellular responses to alkylation chemotherapy. The ability to measure cellular rates of alkylation damage repair by the BER pathway is critically important for better understanding of the fundamental processes involved in carcinogenesis, and also to advance development of new therapeutic strategies. Methods for assessing the rates of alkylation damage and repair, especially in human cells, are limited, prone to significant variability due to the unstable nature of some of the alkyl adducts, and often rely on indirect measurements of BER activity. Here, we report a highly reproducible and quantitative, cell-based assay, named alk-BER (alkylation Base Excision Repair) for measuring rates of BER following alkylation DNA damage. The alk-BER assay involves specific detection of methyl DNA adducts (7-methyl guanine and 3-methyl adenine) directly in genomic DNA. The assay has been developed and adapted to measure the activity of BER in fungal model systems and human cell lines. Considering the specificity and conserved nature of BER enzymes, the assay can be adapted to virtually any type of cultured cells. Alk-BER offers a cost efficient and reliable method that can effectively complement existing approaches to advance integrative research on mechanisms of alkylation DNA damage and repair.

Effect of N7-methylation on base pairing patterns of guanine: a DFT study.

In this paper, we aim to determine whether the N7-methylation can influence the base pairing properties of guanine by promoting the formation of guanine enol-tautomers. The keto- to -enol-tautomerization of N7-methylguanine (N7mG) and its base pairing patterns with all the canonical DNA bases have been investigated at the M06-2X/6-311+G(d,p) level of density functional theory. The barrier free energy calculations reveal that N7-methylation does not promote the keto- to enol- tautomerization of guanine. The Watson-Crick-like enol-N7mG:T1 or enol-N7mG:T2 base pair similar to what is observed experimentally is found to be energetically more stable than the keto-N7mG:T base pairs. However, the keto-N7mG:C1 which is structurally similar to the canonical G:C base pair is the most stable base pair among all the base pairs studied here. Thus, our calculations predict that N7mG would pair preferably with cytosine during DNA replication but there is also a probability that it can cause mutation through mispairing with thymine, in agreement with experimental observations.