This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cytosine methylation (www.ks.uiuc.edu/Research/methylation/) is a chemicalmodification on DNA, which involves replacing a hydrogen atom by a methylgroup at the 5'position in cytosine. Methylation of DNA is one of the most importantmechanisms in epigenetics. Without changing the sequence of DNA, methylationcan alter the expression levels of genes [1]. The physical mechanism underlyingmethylation is presently under intense study, yet current measurement methods formethylation profiles are still lacking. Previous experiments suggest that methylationcan affect DNA properties by changing its structure or its dynamics [2, 3].The Resource is working in close collaboration with Electrical Engineer (G. Timp)and Biological Physicist (H. Gaub), both of whom are experts in single moleculesensors, to elucidate the structural and dynamic properties of methylated DNA aswell as develop new detection methods for DNA methylation. Two atomic scalemodeling experiments were carried out to complement two single molecule experiments:(i) detecting 5'-methylcytosines on DNA with synthetic nanopores and (ii)differential double strand rupture measurements. For study (i), the effect of methylationof DNA was studied computationally by driving negatively charged DNAmolecules through a synthetic nanopore by means of electric fields [4?6]. Simulationsrevealed a difference in nanopore permeation between methylated DNA andun-methylated DNA [7]. In study (ii), simulations of stretching and unzipping doublestrands of methylated and un-methylated DNA were carried out;they showedthat methylated DNA is more stable than un-methylated DNA. Both sets of simulationsnot only agreed well with their respective experimental observations, butalso showed in atomic level detail the reasons for the differences in the mechanicalproperties of methylated and un-methylated DNA.In study (i), experimental measurements showed that the voltage threshold for permeationthrough a nanopore of methylated DNA is lower than that of un-methylatedDNA [7]. Consistent with this, simulations showed a significant difference betweennanopore translocation speeds of methylated and un-methylated DNA at a 4 V bias;these were 1.0 nm/ns and 0.8 nm/ns, respectively. This demonstrates that methylatedDNA passes through the nanopore more readily than un-methylated DNA.These simulations also revealed that the structure of DNA inside the nanopore ismore ordered for methylated than for un-methylated DNA [7]. In study (ii), thesimulations revealed that methylated DNA develops less faults in stretched, i.e., Sform,double strands than un-methylated DNA;this difference results from doublestrands of methylated DNA being harder to separate than those of un-methylatedDNA. These studies suggest that methylation stabilizes DNA mechanically, e.g.,rendering DNA less prone to structural fluctuations. A reduction in structural fluctuationscould readily translate into different transcription levels. The relationshipbetween stability and transcription level is an important insight into this fundamentalmechanism of epigenetics.BIBLIOGRAPHY[1] R. M. Brena, T. H.-M. Huang, and C. Plass. Toward a human epigenome. Nat.Genet., 38:1359?1360, 2006.[2] K. B. Geahigan, G. A. Meints, M. E. Hatcher, J. Orban, and G. P. Drobny. Thedynamic impact of cpg methylation in dna. Biochemistry, 39:4939?4946, 2000.[3] S. Derreumaux, M. Chaoui, G. Tevanian, and S. Fermandjian. Impact of CpG methylationon structure, dynamics and solvation of cAMP DNA responsive element. Nucl.Acids Res., 29:2314?2326, 2001.[4] A. Meller and D. Branton. Single molecule measurements of DNA transport througha nanopore. Electrophoresis, 23:2583?2591, 2002.[5] A. Aksimentiev, J. B. Heng, G. Timp, and K. Schulten. Microscopic kinetics of DNAtranslocation through synthetic nanopores. Biophys. J., 87:2086?2097, 2004.[6] R. F. Service. The race for the $1000 genome. Science, 311:1544?1546, 2006.[7] U. M. Mirsaidov, W. Timp, X. Zou, V. Dimitrov, K. Schulten, A. P. Feinberg, andG. Timp. Nanoelectromechanics of methylated DNA in a synthetic nanopore. Biophys.J., 96:L32?L34, 2009.
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