Non-methylated DNA Research Articles

Cytosine methylation of mitochondrial DNA at CpG sequences impacts transcription factor A DNA binding and transcription

In eukaryotes, cytosine methylation of nuclear DNA at CpG sequences (5mCpG) regulates epigenetic inheritance through alterations in chromatin structure. However, mitochondria lack nucleosomal chromatin, therefore the molecular mechanisms by which 5mCpG influences mitochondria must be different and are as yet unknown. Mitochondrial Transcription Factor A (TFAM) is both the primary DNA-compacting protein in the mitochondrial DNA (mtDNA) nucleoid and a transcription-initiation factor. TFAM must encounter hundreds of CpGs in mtDNA, so the occurrence of 5mCpG has the potential to impact TFAM-DNA recognition. We used biophysical approaches to determine whether 5mCpG alters any TFAM-dependent activities. 5mCpG in the heavy strand promoter (HSP1) increased the binding affinity of TFAM and induced TFAM multimerization with increased cooperativity compared to nonmethylated DNA. However, 5mCpG had no apparent effect on TFAM-dependent DNA compaction. Additionally, 5mCpG had a clear and context-dependent effect on transcription initiating from the three mitochondrial promoters. Taken together, our findings demonstrate that 5mCpG in the mitochondrial promoter region does impact TFAM-dependent activities in vitro.

Voltammetric immunosensor for E-cadherin promoter DNA methylation using a Fe3O4-citric acid nanocomposite and a screen-printed carbon electrode modified with poly(vinyl alcohol) and reduced graphene oxide.

Silencing of tumor suppressor genes (E-cadherin) by promoter DNA methylation may lead to the development of invasive phenotypes in epithelial tissues. The authors describe an electrochemical nanobiosensor for early detection and screening of circulating methylated DNA as a biomarker for cancers. First, the antibody against5-methylcytosine was physically immobilized onto modifiedwith reduced graphene oxide andpolyvinylalcohol. In the next step, methylated target DNA in samples was hybridized with ssDNA probe conjugated to Fe3O4-citric acid nanocomposites and placed on the modified electrode. Then, the hexacyanoferrateredox systemwas added and electron transfer recorded. Cyclic voltammetry and electrochemical impedance spectroscopy showed that the modification process was well accomplished. Quantitative measurement of E-cadherin DNA promoter methylation was performed using differential pulse voltammetry. The electrochemical analysis achieved in the presence and absence of nonmethylated DNA mixed with samples indicated the high specificity and selectivity in methylation analysis using this system. With the linear range of concentration from 1 × 10-4ng.mL-1 to 20ng.mL-1 and the detection limit of 9 × 10-5ng.mL-1, this method represents a promising approach for analysis of other biomarkers. Graphical abstract A label free electrochemical nanobiosensor was constructed for detection of methylated circulating cell-free DNA using screen-printed carbon electrode (SPCE) modified with reduced graphene oxide (rGO) and polyvinylalcohol (PVA).

P152R Mutation Within MeCP2 Can Cause Loss of DNA-Binding Selectivity.

MeCP2 is a protein highly expressed in the brain that participates in the genetic expression and RNA splicing regulation. MeCP2 binds preferably to methylated DNA and other nuclear corepressors to alter chromatin. MECP2 gene mutations can cause rett syndrome (RTT), a severe neurological disorder that affects around one in ten thousand girls. In this paper, Molecular Dynamics (MD) simulations were performed to scrutinize how the MeCP2 P152R mutation influences the protein binding to DNA. Also, the Umbrella Sampling technique was used to obtain the potential mean forces (PMFs) of both wild-type and mutated MeCP2 Methyl-CpG-binding domain (MBD) binding to both non-methylated and methylated DNA. P152R is a common missense mutation in MBD associated with RTT; however, there are no studies that explain how it causes protein dysfunction. The results from this study hypothesize that P152R mutation leads to MBD binding more strongly to DNA, while selectively decreasing binding affinity to methylated DNA. These provide an explanation for previous not conclusive experimental results regarding the mechanism of how this mutation affects the binding of the protein to DNA, and subsequently its effects on RTT. Furthermore, the results of this research-in-progress can be used as the basis for further investigations into the molecular basis of RTT and to potentially reveal a target for therapy in the future.

Coordinated Dialogue between UHRF1 and DNMT1 to Ensure Faithful Inheritance of Methylated DNA Patterns

DNA methylation, catalyzed by DNA methyltransferases (DNMTs), is an epigenetic mark that needs to be faithfully replicated during mitosis in order to maintain cell phenotype during successive cell divisions. This epigenetic mark is located on the 5′-carbon of the cytosine mainly within cytosine–phosphate–guanine (CpG) dinucleotides. DNA methylation is asymmetrically positioned on both DNA strands, temporarily generating a hemi-methylated state after DNA replication. Hemi-methylation is a particular status of DNA that is recognized by ubiquitin-like containing plant homeodomain (PHD) and really interesting new gene (RING) finger domains 1 (UHRF1) through its SET- (Su(var)3-9, Enhancer-of-zeste and Trithorax) and RING-associated (SRA) domain. This interaction is considered to be involved in the recruitment of DNMT1 to chromatin in order to methylate the adequate cytosine on the newly synthetized DNA strand. The UHRF1/DNMT1 tandem plays a pivotal role in the inheritance of DNA methylation patterns, but the fine-tuning mechanism remains a mystery. Indeed, because DNMT1 experiences difficulties in finding the cytosine to be methylated, it requires the help of a guide, i.e., of UHRF1, which exhibits higher affinity for hemi-methylated DNA vs. non-methylated DNA. Two models of the UHRF1/DNMT1 dialogue were suggested to explain how DNMT1 is recruited to chromatin: (i) an indirect communication via histone H3 ubiquitination, and (ii) a direct interaction of UHRF1 with DNMT1. In the present review, these two models are discussed, and we try to show that they are compatible with each other.

Allelic and epigenetic DNA variation in relation to F1 heterosis manifestation in F1 hybrids of Capsicum annuum L.

Managing F1heterosis is one of the major objectives in hybrid crop breeding programs. The classical theory considers the heterozygosity in F1hybrids to be the main factor contributing to heterosis and therefore presumes a linear relationship between the value of genetic polymorphisms in parental lines and the heterotic response of their F1offspring. Therefore, the genetic diversity information is viewed as a tool for selection of promising cross-combinations, but results published by different researchers are inconsistent. In this work, we studied the contributions of structural and nonstructural DNA polymorphisms to F1heterosis manifestation. We used SSR and methyl-sensitive AFLP (MSAP with HpaII and MspI izoshisomers) protocols for obtaining specific patterns for heterotic and nonheterotic F1hybrids of sweet pepper (Capsicum annuum L.) from a Belarusian breeding program. We found out that a certain portion of heterosis for yield-related traits might be explained by the polymorphism revealed by SSR analysis. According to our data, the total number of polymorphic SSR loci and the ratio of polymorphic and nonpolymorphic loci demonstrate a significant predictive value and can serve as additional prognostic criteria for the selection of promising cross-combinations. From the MSAP assay, we found a relationship between heterosis and the numbers of methylated and nonmethylated DNA loci for yield traits. Our results indicate that cross-hybridization may favor epiallelic modifications in F1hybrids, presumably responsible for heterosis. Thus, epigenetic DNA variation may explain the absence of a linear relationship between the level of structural DNA divergence and F1heterosis, as well as the manifestation of heterosis in crosses of related (genetically similar) accessions.

Discrimination of methylated and nonmethylated region of a colorectal cancer related promoter using fluorescence enhancement of gold nanocluster at intrastrand of a 9C-loop

Among epigenetic modifications of DNA, methylation of cytosine at its carbon 5 is the most common mark that is usually associated with gene silencing in human. Determining whether a particular DNA molecule is methylated or not, is an indispensable task in many epigenetic investigations. Presenting detection methods with less labor-intensive and time-consuming procedures has substantial value. Here a facile method based on gold nanocluster (AuNCs) fluorescence enhancement is presented. Target sequences were selected from Sept9 promoter region as its hypermethylation is demonstrated as a reliable biomarker of colorectal cancer. DNA probe was complementary to a 25 nucleotide of the target region and possessed 9 additional cytosines in the middle to allow the formation of AuNCs. Probe-AuNCs strands were first hybridized with methylated and non-methylated targets separately, and then their fluorescence intensities were recorded. Fluorescence intensity was higher with methylated targets than non-methylated one. Applying silver ions reversed the situation and fluorescence intensities of non-methylated DNA got higher than methylated one.

PITX2 DNA-methylation as the first clinically validated predictive marker for anthracycline based chemotherapy in high-risk breast cancer patients

International guidelines recommend anthracycline-based chemotherapy for high-risk breast cancer patients as the standard-of-care, but not all patients do equally benefit from such a chemotherapy. The novel therascreen® PITX2 RGQ PCR assay is a quantitative in vitro methylation-specific real-time PCR test (qMSP), intended for the determination of the percent methylation ratio (PMR) in the pituitary homeobox 2 (PITX2) promoter 2 expressed by primary breast cancer tumor tissue (FFPE-material). After bisulfite exposure of extracted DNA to distinguish between methylated and non-methylated PITX2 DNA, the percent methylation ratio (PMR) of the PITX2 gene promoter 2 is quantified by qMSP. The PMR obtained does provide information to the treating physician about whether a patient is likely to respond to anthracycline-based chemotherapy. Data will be presented for 205 high-risk lymph node-positive, estrogen receptor-positive, HER2-negative breast cancer patients, treated with adjuvant anthracycline-based chemotherapy. The PITX2 pre-defined cut-off value of PMR = 12 demonstrated a statistically significant differentiation between low- and high-risk breast cancer patient for the primary endpoint DFS, also for patients treated with endocrine therapy in addition to anthracycline-based adjuvant therapy. High-risk lymph node-positive, estrogen-receptor-positive, HER2-negative breast cancer patients, with PITX2 methylation defined as low (PMR ≤ 12), are sufficiently treated with anthracycline-based chemotherapy, irrespective if treated with additional tamoxifen. High-risk lymph node-positive, estrogen receptor-positive, HER2-negative breast cancer patients with PITX2 methylation defined as high (PMR > 12) should avoid anthracycline-based chemotherapy. These patients are recommended to switch to other chemotherapy regimens, since with this PITX2-characteristics, the patient has a lower probability to respond to anthracycline-based chemotherapy.

Abstract B028: Comparative assessment of cellular functions for the ZBTB methyl-CpG binding proteins in prostate cancer

Abstract It is becoming increasingly evident that DNA methylation patterns are misappropriated in a number of cancers. This leads to downstream alterations in transcription that afford a survival advantage for the disease state cells. In many cases, DNA methylation directs gene expression through the recruitment of methyl-CpG binding proteins (MBPs). These specialized factors read the methylated DNA signal and translate this into recruitment of chromatin remodeling complexes. The ZBTB family of MBPs, consisting of ZBTB33, ZBTB4, and ZBTB38, are unique among MBPs in that they utilize a single protein domain to specifically target both methylated and sequence-specific nonmethylated DNA sites. This bimodality in DNA recognition affords these proteins the ability to effect either transcriptional repression or activation depending on the cellular context. Further, there is mounting evidence that overexpression of ZBTB MBPs and their subsequent transcriptional activities are directly associated with cancer, though the mechanism by which each protein functions to maintain and/or progress the disease state remains to be elucidated. Of particular note, ZBTB33 overexpression has been correlated with tumorigenicity in prostate cancer (PCa). In these studies we sought to begin more thoroughly characterizing the role of ZBTB33 in PCa, and simultaneously evaluating whether ZBTB38 and ZBTB4 may also participate in this disease. Utilizing both cell line models and patient PCa tumors, we demonstrate that protein levels for all three ZBTB MBPs increase in the cancerous state relative to normal cells/tissues. Notably, the expression levels of ZBTB4 appear to peak at earlier stage disease and decrease at later. To begin delineating the biologic pathways mediated by each ZBTB MBP, we evaluated global transcriptome (RNA-seq) and phenotypic alterations after systematic ZBTB MBP depletion in two phenotypically distinct PCa cell line models. Specifically, we evaluated the impact of each ZBTB MBP on cell proliferation, apoptosis, and migration. In short, we determined that cellular phenotypic response to depletion of the three ZBTB MBPs appears highly variable between proteins as well as cell line model. Further, comparative RNA-seq after depletion of each ZBTB MBP demonstrates that consistent with the phenotypic analyses, each ZBTB MBP appears to mediate protein-specific transcriptional responses. These findings suggest that these proteins may function in parallel within PCa. Combined, these studies represent the first comprehensive analysis of all three ZBTB MBPs in the same disease context and emphasize the potential for these proteins as novel therapeutic targets. Citation Format: Amir Pozner, Aneesa T. Al-Soodani, Sheryl Tripp, Justin Caron, Daniel J. Albertson, Bethany A. Buck-Koehntop. Comparative assessment of cellular functions for the ZBTB methyl-CpG binding proteins in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B028.

Structural basis for the ability of MBD domains to bind methyl-CG and TG sites in DNA

Cytosine methylation is a well-characterized epigenetic mark and occurs at both CG and non-CG sites in DNA. Both methylated CG (mCG)- and mCH (H = A, C, or T)-containing DNAs, especially mCAC-containing DNAs, are recognized by methyl-CpG-binding protein 2 (MeCP2) to regulate gene expression in neuron development. However, the molecular mechanism involved in the binding of methyl-CpG-binding domain (MBD) of MeCP2 to these different DNA motifs is unclear. Here, we systematically characterized the DNA-binding selectivities of the MBD domains in MeCP2 and MBD1-4 with isothermal titration calorimetry-based binding assays, mutagenesis studies, and X-ray crystallography. We found that the MBD domains of MeCP2 and MBD1-4 bind mCG-containing DNAs independently of the sequence identity outside the mCG dinucleotide. Moreover, some MBD domains bound to both methylated and unmethylated CA dinucleotide-containing DNAs, with a preference for the CAC sequence motif. We also found that the MBD domains bind to mCA or nonmethylated CA DNA by recognizing the complementary TG dinucleotide, which is consistent with an overlooked ligand of MeCP2, i.e. the matrix/scaffold attachment regions (MARs/SARs) with a consensus sequence of 5'-GGTGT-3' that was identified in early 1990s. Our results also explain why MeCP2 exhibits similar binding affinity to both mCA- and hmCA-containing dsDNAs. In summary, our results suggest that in addition to mCG sites, unmethylated CA or TG sites also serve as DNA-binding sites for MeCP2 and other MBD-containing proteins. This discovery expands the genome-wide activity of MBD-containing proteins in gene regulation.

Leukocyte Counts Based on DNA Methylation at Individual Cytosines.

White blood cell counts are routinely measured with automated hematology analyzers, by flow cytometry, or by manual counting. Here, we introduce an alternative approach based on DNA methylation (DNAm) at individual CG dinucleotides (CpGs). We identified candidate CpGs that were nonmethylated in specific leukocyte subsets. DNAm levels (ranging from 0% to 100%) were analyzed by pyrosequencing and implemented into deconvolution algorithms to determine the relative composition of leukocytes. For absolute quantification of cell numbers, samples were supplemented with a nonmethylated reference DNA. Conventional blood counts correlated with DNAm at individual CpGs for granulocytes (r = -0.91), lymphocytes (r = -0.91), monocytes (r = -0.74), natural killer (NK) cells (r = -0.30), T cells (r = -0.73), CD4+ T cells (r = -0.41), CD8+ T cells (r = -0.88), and B cells (r = -0.66). Combination of these DNAm measurements into the "Epi-Blood-Count" provided similar precision as conventional methods in various independent validation sets. The method was also applicable to blood samples that were stored at 4 °C for 7 days or at -20 °C for 3 months. Furthermore, absolute cell numbers could be determined in frozen blood samples upon addition of a reference DNA, and the results correlated with measurements of automated analyzers in fresh aliquots (r = 0.84). White blood cell counts can be reliably determined by site-specific DNAm analysis. This approach is applicable to very small blood volumes and frozen samples, and it allows for more standardized and cost-effective analysis in clinical application.

GDNA extraction yield and methylation status of blood samples are affected by long-term storage conditions.

Epigenetics is believed to provide great chances for a better understanding of the development and treatment of many diseases where the analysis of genomic DNA has so far failed to provide conclusive answers. Methylcytosine is a frequently used quantitative marker of epigenetic studies. Since immediate analysis of sampled material is in most cases not possible, storage time and conditions are critical aspects regarding the quality of genomic DNA and reliability of analysis. Blood is frequently used for such analyses. We, therefore, collected blood samples of ten volunteers and stored them under various conditions for ten months: -70°C, -20°C, 2–8°C and room temperature. An additional aliquot was frozen at -70°C and thawed once a week at room temperature. We then compared the DNA extraction yields and methylation status in relation to storage time and conditions. We found significantly lower DNA extraction yields (up to -97.45%; p ≤ 0.001) as well as significantly higher methylation levels after ten months of storage (up to +42.0%; p ≤ 0.001). These results suggest that storage time has an important influence on DNA analyses of blood samples for all storage conditions. This might be due to differences in stability of methylated and non-methylated DNA. Our study indicates that storage conditions and time may be a critical factor for epigenetic methylation studies and require rigorous validation. For reliable analyses we, therefore, recommend to perform epigenetic analysis directly after sample collection.

In vivo analysis of circulating cell-free DNA release and degradation

Cell-free DNA (cfDNA) was first detected in human plasma in the 1940s, but the knowledge on its regulation and rate of release is incomplete. CfDNA can originate from both normal and tumour cells. Our aims were to investigate the rate of cfDNA's release in SHO mice/HT-29 colorectal adenocarcinoma cell line xenograft model and to define the decay of methylated and non-methylated DNA fragments in C57BL/6 bloodstream. SHO mice were xenografted with human HT-29 cells, than blood samples were collected over 2 months. CfDNA was isolated, then quantified by real-time PCR with highly specific genomic and mitochondrial human and mouse primer sets. This method permitted to define the ratio of human/mouse DNA. To assess the degradation rate of cfDNA, 3000 bp sized methylated and non-methylated DNA fragments were injected into healthy and C38 tumour-cell vaccinated C57BL/6 mice's bloodstream. The decay of amplicons was measured with 19 PCR assays. The amount of human DNA until the 2nd week was below the limit of detection. From the third week, a continuous growth was experienced, which reached 18.26% by the 8th week. Moreover, it was found that in healthy animals the non-methylated DNA disappears from the plasma after 6 hours, while the methylated fragment was detectable even after 24 hours. In animals with tumour, both amplicons were detectable after 24 hours. The examination of the role and mechanism of cfDNA shows an increasing level of interest. This work can contribute to a better understanding of the release and degradation of cfDNA. Orv Hetil. 2018; 159(6): 223-233.

Biochemical Identification of Nonmethylated DNA by BioCAP-Seq.

CpG islands are regions of vertebrate genomes that often function as gene regulatory elements and are associated with most gene promoters. CpG island elements usually contain nonmethylated CpG dinucleotides, while the remainder of the genome is pervasively methylated. We developed a biochemical approach called biotinylated CxxC affinity purification (BioCAP) to unbiasedly isolate regions of the genome that contain nonmethylated CpG dinucleotides. The resulting highly pure nonmethylated DNA is easily analyzed by quantitative PCR to interrogate specific loci or via massively parallel sequencing to yield genome-wide profiles.

The SET1 Complex Selects Actively Transcribed Target Genes via Multivalent Interaction with CpG Island Chromatin

SummaryChromatin modifications and the promoter-associated epigenome are important for the regulation of gene expression. However, the mechanisms by which chromatin-modifying complexes are targeted to the appropriate gene promoters in vertebrates and how they influence gene expression have remained poorly defined. Here, using a combination of live-cell imaging and functional genomics, we discover that the vertebrate SET1 complex is targeted to actively transcribed gene promoters through CFP1, which engages in a form of multivalent chromatin reading that involves recognition of non-methylated DNA and histone H3 lysine 4 trimethylation (H3K4me3). CFP1 defines SET1 complex occupancy on chromatin, and its multivalent interactions are required for the SET1 complex to place H3K4me3. In the absence of CFP1, gene expression is perturbed, suggesting that normal targeting and function of the SET1 complex are central to creating an appropriately functioning vertebrate promoter-associated epigenome.

Optimized short digestion protocol for free fetal DNA detection using methylation-dependent markers

Abstract Background: Circulating free DNA (cfDNA) digestion with methylation sensitive restriction enzymes constitutes an important diagnostic tool for differentiating methylated from non-methylated DNA sequences. In the context of pregnancy, this is used to differentiate fetal from maternal DNA. Current protocols are of long duration and use multiple enzymes with different incubation and inactivating temperatures. We describe a short protocol for the digestion of circulating free DNA focused on its future adaptation to miniaturized microfluidic devices based on lab-on-a-chip technology. Methods: cfDNA was extracted from plasma samples of pregnant and non-pregnant women with chemagic Viral NA/gDNA and QIAamp circulating nucleic acids kits. For digestion protocol optimization, different methylation sensitive and insensitive restriction enzymes were used. Detection of RASSF1A, SRY and (β-actin) ACTB sequences was performed by real time polymerase chain reaction (PCR). Results: The digestion protocol is optimized to a 3.5 h one-step protocol using the enzymes BstUI, BstY1 and HhaI resulting in a complete digestion of the hypomethylated maternal RASSF1A with a limit of digestion of 3.65E10 gene copies. Conclusions: This work provides a digestion protocol for cfDNA samples with a combination of temperatures (37 °C and 60 °C) and a protocol length (<4 h) which facilitates its adaptation to miniaturized microfluidic devices based on lab-on-a-chip technology. In this technology, the shorter the duration of the protocol, the greater the rate of success and the less sample evaporation.

Methylated Cytosine Maintains G-Quadruplex Structures during Polymerase Chain Reaction and Contributes to Allelic Dropout.

The promoter of the human imprinted gene MEST is differentially methylated with respect to the parent of origin and contains several non B-DNA motifs that are capable of forming G-quadruplexes. These factors can contribute to a consistent allelic dropout (ADO) of the maternally methylated DNA during polymerase chain reaction (PCR) analysis of such gene regions. Here, we directly investigate the cause of allelic dropout by applying fluorescent techniques to visualize polymerase amplification and arrest during PCR of differentially methylated DNA templates. We demonstrate that polymerase arrest corresponds to previously characterized G-quadruplex-forming motifs at the MEST promoter region and occurs at equivalent sites on both methylated and nonmethylated DNA templates. However, during PCR, polymerase arrest can be observed on the methylated template for several cycles longer than on the nonmethylated template, and this results in an amplification lag and a lower yield of full length amplicons. We demonstrate that this delay in amplification is sufficient to cause complete ADO during PCR, providing a mechanistic basis for the previously observed genotyping error at this locus.

Effect of DNA Methylation on the Velocity of DNA Translocation through a Nanochannel.

Here, we report the effect of DNA methylation on the velocity of DNA translocation through a nanochannel, as determined by measuring differences in translocation velocities between methylated and non-methylated DNA molecules. We found that the velocity of translocation of methylated DNA was faster than that of non-methylated DNA, which we attributed to variation in the coefficients of diffusion and friction with the nanochannel wall, due to the increased molecular weight and stiffness, respectively, of methylated DNA.

Methylation-targeted specificity of the DNA binding proteins R.DpnI and MeCP2 studied by molecular dynamics simulations.

DNA methylation plays a major role in organismal development and the regulation of gene expression. Methylation of cytosine bases and the cellular roles of methylated cytosine in eukaryotes are well established, as well as methylation of adenine bases in bacterial genomes. Still lacking, however, is a general mechanistic understanding, in structural and thermodynamic terms, of how proteins recognize methylated DNA. Toward this aim, we present the results of molecular dynamics simulations, alchemical free energy perturbation, and MM-PBSA calculations to explain the specificity of the R.DpnI enzyme from Streptococcus pneumonia in binding to adenine-methylated DNA with both its catalytic and winged-helix domains. We found that adenine-methylated DNA binds more favorably to the catalytic subunit of R.DpnI (-4kcalmol-1) and to the winged-helix domain (-1.6kcalmol-1) than non-methylated DNA. In particular, N6-adenine methylation is found to enthalpically stabilize binding to R.DpnI. In contrast, C5-cytosine methylation entropically favors complexation by the MBD domain of the human MeCP2 protein with almost no contribution of the binding enthalpy.

Dynamics of Methylated Cytosine Flipping by UHRF1.

DNA methylation patterns, which are critical for gene expression, are replicated by DNA methyltransferase 1 (DNMT1) and ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) proteins. This replication is initiated by the recognition of hemimethylated CpG sites and further flipping of methylated cytosines (mC) by the Set and Ring Associated (SRA) domain of UHRF1. Although crystallography has shed light on the mechanism of mC flipping by SRA, tools are required to monitor in real time how SRA reads DNA and flips the modified nucleobase. To accomplish this aim, we have utilized two distinct fluorescent nucleobase surrogates, 2-thienyl-3-hydroxychromone nucleoside (3HCnt) and thienoguanosine (thG), incorporated at different positions into hemimethylated (HM) and nonmethylated (NM) DNA duplexes. Large fluorescence changes were associated with mC flipping in HM duplexes, showing the outstanding sensitivity of both nucleobase surrogates to the small structural changes accompanying base flipping. Importantly, the nucleobase surrogates marginally affected the structure of the duplex and its affinity for SRA at positions where they were responsive to base flipping, illustrating their promise as nonperturbing probes for monitoring such events. Stopped-flow studies using these two distinct tools revealed the fast kinetics of SRA binding and sliding to NM duplexes, consistent with its reader role. In contrast, the kinetics of mC flipping was found to be much slower in HM duplexes, substantially increasing the lifetime of CpG-bound UHRF1, and thus the probability of recruiting DNMT1 to faithfully duplicate the DNA methylation profile. The fluorescence-based approach using these two different fluorescent nucleoside surrogates advances the mechanistic understanding of the UHRF1/DNMT1 tandem and the development of assays for the identification of base flipping inhibitors.

Insights into Damaged Base Detection by DNA Glycosylases: A Computational Study of AlkD

In order to protect the cellular genome from damage, a wide array of proteins known as DNA repair enzymes must be able to rapidly locate nucleic acid modifications. The first step of the base excision repair pathway is the removal of modified nucleotides, which requires that DNA glycosylases be able to efficiently detect their target. Once located, most DNA glycosylases utilize the base-flipping mechanism to flip damaged nucleobases into a binding pocket on the protein. Recent crystallization studies show that there is at least one exception, AlkD, which repairs methylated DNA without forcing the damaged nucleobase out of its base pair. The unique nature of AlkD allows us to investigate the ways in which DNA glycosylases locate modified DNA. Using microsecond MD simulations, we demonstrate that AlkD is able to stabilize a dramatically contorted DNA structure by promoting localized B-to-A-DNA transitions in the methylated DNA. On the contrary, non-methylated DNA is found to unbind from the putative binding region in order to form a more linear B-DNA type conformation. These observations are further supported by computing the free energy of binding for methylated and non-methylated DNA sequences.