What is DNA Methyl at Ion

8/2/2019 What is DNA Methyl at Ion

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DNA methylation

DNA methylation

What is DNA methylation?

Methylation ofcytosine is acovalentmodificationof DNA, in

whichhydrogen H5of cytosine isreplaced by amethyl group.In mammals, 60% - 90% of all CpGs are methylated. Methylation adds informationnot encoded in the DNA sequence, but it does not interfere with the Watson-Crickpairing of DNA - the methyl group is positioned in the major groove of the DNA. Thepattern of methylation controls protein binding to target sites on DNA, affectingchanges in gene expression and in chromatin organization, often silencing genes,which physiologically orchestrates processes like differentiation, and pathologicallyleads to cancer [reference see publications below].

Biological functions of DNA methylation

Transcriptional gene silencingGenome stabilityChromatin compactionGenome defenseSuppression of homologous recombination between repeatsX chromosome inactivation (females)

(Nature Genetics 38:1359 (2006))

H o m e S o f t w a r e R e s e a r c h P u b l i c a t i o n s I n s t r u c t i o n

M e d i a F a c i l i t i e s A b o u t U s

Page 1 of 15DNA methylation

1/25/2012http://www.ks.uiuc.edu/Research/methylation/


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Same Genes but a Different Kink in the Tail. PLoS Biol 1:3

(2003)

(Photograph courtesy of Emma Whitelaw, University ofSydney, Australia.)

Hypermethylatio

Using nanopore to detect DNA methylation

We have reported measurements and simulations of the permeation of methylatedDNA through a synthetic nanopore, using an electric field to force single moleculesto translocate across the membrane through the pore (see also our nanoporesite). We observe a voltage threshold for permeation of methylated DNA through a< 2nm diameter pore, which we attribute to the stretching transition of DNA,occurring in such pore induced by the voltage gradient; the degree of stretching candiffer by >1V/20nm depending on the methylation level, but does not dependsensitively on DNA sequence. The threshold for squeezing unmethylated DNA

through 1.8 nm pore is about 3.6 V while fully-methylated DNA shows a threshold2.7 V. In MD simulation, we recognized a significant difference between thetranslocation speeds of methylated and unmethylated DNA, namely, speeds of 1.0nm/ns and 0.8 nm/ns, respectively. The velocity difference implies that methylatedDNA passes the pore more readily than unmethylated DNA, which indeed isconsistent with the threshold voltage difference measured. Conformations of theDNAs are shown in the figure below. One can recognize that both translocatingDNAs exhibit a B-form structure. However, methylated DNA is considerably moreordered and adheres more closely to the ideal B-form than does unmethylatedDNA. More detail can be found here.




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Using nanopore to detect DNA methylati

DNA methylation affects strand separation

The role of cytosine methylation in epigenetics isgenerally understood to involve effects of DNAmethylation on chromatin structure or on the action oftranscription factors, either one effect controlling in along-time manner gene expression levels and,thereby, cell differentiation. The small effect of

cytosinemethylation

on DNA

thermodynamic properties let one to believe that a change of DNA physical




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properties by themselves, i.e., not ones mediated by the nucleosome andtranscription factors, do not need to be considered for a role in epigenetics. Ourstudy shows now that a role of methylated DNA physical properties needs to bereconsidered as a direct epigenetic control factor. We found through novel andextremely extensive force measurements comparing non-methylated and variousmethylated DNAs that methylation affects the propensity for mechanical DNAstrand separation to a significant degree.

Determination of the mechanical stability of methylated DNA by molecular

force assay measurements. Measurements stretching DNA mechanically to thepoint of strand separation were carried out with a new chip technology, molecularforce assay (MFA), that permits one to stretch and rupture apart thousands of DNAdouble strands organized by methylation type on spots on the chip with afluorescent signal readout. The experimental setup of the MFA at a molecular levelconsists of molecular force probes (MFPs). The MFPs are composed of two DNAduplexes, a target duplex 1 2 and a reference duplex 2 3, which are coupled inseries and connected between two surfaces. Here, the target DNA duplex is 20 bplong and contains zero (nDNA), one (mC-1c-DNA) or three (mC-3-DNA) 5-methylcytosines (mC) per strand, while the reference duplex is the same for all

three different MFPs. About 104 MFPs per m2 are anchored in parallel between aglass slide (lower surface) and a PDMS stamp (upper surface). The different MFPsare immobilized as well separated spots on the glass substrate (see figure below).

During separation of the two surfaces, a force builds up gradually in the MFPs untilone of their two DNA duplexes ruptures, either the target duplex or the referenceduplex. After separation, the ratio of ruptured target to reference duplexes is readout via the fluorescence signal of the MFPs on the lower surface and analyzed toobtain the normalized fluorescence (NF). The NF is defined as the ratio of brokenreference bonds to the total amount of MFPs that have been under load. Thus theNF is a measure for the relative mechanical stability between target and referenceDNA duplexes of a MFP: a higher NF denotes a mechanical stability of the target

duplex elevated over that of the reference duplex. The difference in NF reflects asignificant difference in mean rupture force between nDNA, mC-1c-DNA and mC-3-DNA.




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Molecular force assay

Determination of potential width and dissociation constant. To furtherinvestigate the differences in strand separation of methylated and non-methylatedDNA, single molecule force spectroscopy rupturing the DNA double-strand wasperformed by AFM. In all experiments one single-stranded DNA (ssDNA) wasbound with a poly-ethyleneglycol (PEG) spacer to the cantilever tip of the AFM anda second ssDNA was immobilized with a PEG spacer on a glass substrate. Whilethe tip of the AFM approached the surface, the ssDNA on the tip and the ssDNA on

the glass substrate could form a 20 bp duplex. The tip was then retracted from thesurface and the DNA duplex was loaded with an increasing force until it finallyruptured (see figure below). The same sequence of the DNA duplex was used as inthe MFA experiments with zero (nDNA), one (mC-1c-DNA) or three (mC-3-DNA) 5'-methylcytosines (mC) per strand. Complementing the MFA experiments, the AFMexperiments reveal an elevated mechanical stability for mC-3-DNA and adecreased stability for mC-1c-DNA in comparison to nDNA.




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Simulation system

Atomic force microscopy

MD simulations of DNA strand separation. In our simulations, non-methylatedDNA (nDNA), center-methylated DNA (cDNA) and fully-methylated DNA (fDNA)

were subjected to a stretching force directed along the helical axis. Figure belowshows force-extension curves for nDNA, cDNA and fDNA stretched with a pullingvelocity of 1 /ns. The force-extension profile exhibits in each case a clear peakfollowed by a rapid force decrease. The force-extension curve of cDNA exhibits aminor and a major force peak. Examination of the respective simulation trajectoryrevealed that some flipped-out bases of the two already separated strands of cDNAstacked on each other after initial, partial separation (minor peak). As a result, astronger force (major peak) was needed to completely separate the DNA strands.Before DNA reaches an extension of 13.8 nm, the force-extension curves of fDNA,




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cDNA and nDNA are indistinguishable. However, upon further extension the forceneeded to extend methylated DNA is larger than the force needed to extend non-methylated DNA, indicating that methylation affects the late-stage of force-inducedDNA strand separation.

MD simulations

We characterized the strand separation pathway through the 5' end - 5' enddistance (extension), stacking energy of DNA bases, and intermediate DNAgeometries. Because of thermal fluctuation, some bases keep floating out of thezipper-like packing, and leave holes in the zipper, referred to as ``bubbles". Theamount of developed bubbles influences the stability of DNA against strandseparation. As can be seen in the figure below, the density of bubbles, on average,is lower for DNA with more methylation sites, which is consistent with the result thatfDNA requires a stronger rupture force than nDNA in our simulations.




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Strand separation pathway of stretched DNA

To investigate the strand separation of nDNA and fDNA under conditions closer tothe experimental loading force, we carried out SMD simulations at 200 pN constantforce on two DNAs for 90 ns. The figure below shows that under 200 pN stretching,

nDNA and fDNA extend along the backbone axis as the DNA increases over 90 nsits extension values. Monitoring stacking energy and length of DNA, one candistinguish two different conformational spaces occupied by nDNA and fDNA.Consistent with the strand separation pathways obtained from fast-pulling (10 /ns)SMD simulations, fDNA remains more compact than nDNA does. From theconformations shown in the inset of Figure below, one can see that zipper-likefDNA develops less bubbles than zipper-like nDNA does.




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Stacking energy vs. extension data from two 90-ns-long constant

force simulations pulling DNA

Methyl-CpG binding domain (MBD) proteinsrecognize methylated DNA

MBD protein family. At present, five proteins with mCpG-binding motifs havebeen identified in the MBD family, namely MBD1, MBD2, MBD3, MBD4 andMeCP2. All MBD proteins share a similar MBD of about 75 amino-acid residues.Sequence alignment of MBD proteins reveals several highly conserved residues onthe interface between MBD and methylated DNA (mDNA). Figure below shows thatthe most conserved region in the primary structure of the MBD is located betweenresidues ARG22 and LYS46, including loop L1, strand 3, strand 4 and loop L2.The conserved residues of the MBD form the interfacial surface contacting mDNA

and are responsible for recognition of the methyl-CpG steps.




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Sequence alignment of five methyl-CpG binding domain (MBD) proteins from human

MBD proteins bound to mDNA. The NMR structures of MBD1, MBD2 and the X-ray structure of MeCP2 proteins bound to the mDNA provide useful information onhow residues in the MBD contact mDNA. Figure below illustrates the MBDinteraction with the mCpG steps in the major groove of mDNA. The methyl groupsof methyl-cytosine are buried in the mDNA-MBD interface. The principal mCpGbinding interface composed of hydrophilic and hydrophobic residues involves twotypes of interaction with mCpG steps: the polar atoms in MBD form hydrogen bondswith the polar atoms in the DNA, while the non-polar atoms in MBD form ahydrophobic patch around the methyl groups.




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MBD proteins binding to mDNA. Shown are the structures of (a) MBD1-mDNA, (b) M

Molecular dynamics simulation. Structures of MBD1-mDNA, MBD2-mDNA andMeCP2-mDNA complexes reveal that the conserved residues ARG22 and ARG44form hydrogen bonds with guanines in the mCpG steps. To investigate the role ofARG22 and ARG44 in recognizing the mCpG steps, we conducted a 100-nsmolecular dynamics simulation of the NMR structure of the human MBD1-DNAcomplex. Simulations showed that the spatial configurations of mCYT106-ARG22-GUA107 and mCYT118- ARG44-GUA119 adopt a so-called stair motif. Similar stairmotifs are formed and remain stable at the MBD2-mDNA and MeCP2-mDNAinterface.




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Stair motifs in the interface of MBD1-mD

Mutational studies in which mCYT106 and mCYT118 were substituted by nCYT106and nCYT118 (nCYT denotes non-methylated cytosine) revealed that MBD1 doesnot bind to non-methylated DNA, indicating that the 5-methyl groups on the methyl-cytosines are critical for the formation of the stair motif in MBD-mDNA bindinginterfaces. The influence of methylation on the stability of the stair motif wasinvestigated through a 30-ns MD simulation for the MBD1-nDNA complex, wherethe mCYT106 and mCYT118 nucleotides were mutated into two non-methylatedcytosines. Our resutls showed that the interactions between ARG and mCYT are

more stable than those between ARG and nCYT, as methylation increases thecontact area between mDNA and the MBD.




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Effect of methylation on the MBD1-DNA contact area.

Ab initio quantum chemistry calculations. We performed quantum chemistrycalculations of the interaction energy in the mCYT-ARG-GUA and nCYT-ARG-GUAstair motifs in order to determine the impact of the methyl group on the stair motifstabilization. QM calculations show a stabilization effect of cytosine methylation onthe studied system, see figure below. The blue line (squares) shows the interactionenergy calculated for the mCYT106-ARG22-GUA107 stair motif, the red line(triangles) shows the interaction energy calculated for the mCYT118-ARG44-GUA119 stair motif, while the green line (dots) shows the combined energydifference.




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Influence of cytosine methylation on MBD1 binding energy.

Free-energy contributions of DNA methylation to MBD1-DNA complex

stabilization. The protein-DNA binding affinity is affected by different interactions.In order to account for the total effect of methylation on the stability of the MBD-DNA complex, we performed FEP calculations to characterize the free-energycontribution of the methyl group to the MBD-DNA complex. The BAR (see Methods)free-energy difference for the free DNA was determined to be G1 = 54.9 0.0kcal/mol, with -0.12 kcal/mol hysteresis between forward and backward transition;for the MBD-DNA complex, the free-energy difference was determined to be G2 =53.7 0.1 kcal/mol, with 0.2 kcal/mol hysteresis between forward and backwardtransition. Following thermodynamic cycle shown below, one can obtain G = -1.2 0.1 kcal/mol, hence, indicating that methylation makes a favorable contribution to

the binding free energy.

Thermodynamic cycle describing DNA methylation in the DNA-MBD complex.

Publications

Recognition of methylated DNA through methyl-CpG binding domain

proteins Xueqing Zou, Wen Ma, Ilia Solov'yov, Christophe Chipot and KlausSchulten Nucleic Acids Research, doi: 10.1093/nar/gkr1057




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Cytosine methylation alters DNA mechanical properties Philip M.D.Severin*, Xueqing Zou*, Hermann E. Gaub, and Klaus Schulten NucleicAcids Research, 49:8740-8751, 2011

Nanoelectromechanics of Methylated DNA in a Synthetic Nanopore U.Mirsaidov, W. Timp, X. Zou, V. Dimitrov, K. Schulten, A. P. Feinberg, and G.Timp Biophysical Journal, 96:L32-L34, 2009

* Equal contribution authors

Investigators

Simulations

Xueqing ZouWen MaIlia Solov'yovChristophe ChipotKlaus Schulten

ExperimentPhilip M.D. SeverinHermann E. GaubUtkur M. MirsaidovGreg Timp

Related TCB Group Projects

Detecting DNA Orientation with a NanoporeTranslocation of DNA through Synthetic Nanopores

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Beckman Institute for Advanced Science and Technology // National Institutes of Health // National Science Foundation //

Physics, Computer Science, and Biophysics at UIUC

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What is DNA Methyl at Ion

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