Identifying Protein–(Hydroxy)Methylated DNA Using Quantitative Interaction Proteomics

<div> <div> <div> <p>Partition coefficients serve in various areas as pharmacology and environmental sciences to predict the hydrophobicity of different substances. Recently, they have been also used to address the accuracy of force fields for various organic compounds and specifically the methylated DNA bases. In this study atomic charges were derived by different partitioning methods (Hirshfeld and Minimal Basis Iterative Stockholder) directly from the electron density obtained by electronic structure calculations in vac- uum, with an implicit solvation model or with explicit solvation taking the dynamics of the solute and the solvent into account. To test the ability of these charges to describe electrostatic interactions in force fields for condensed phases the original atomic charges of the AMBER99 force field were replaced with the new atomic charges and combined with different solvent models to obtain the hydration and chloroform solvation free energies by molecular dynamics simulations. Chloroform-water partition coefficients derived from the obtained free energies were compared to experimental and previously reported values obtained with the GAFF or the AMBER-99 force field. The results show that good agreement with experimental data is obtained when the polarization of the electron density by the solvent has been taken into account deriving the atomic charges of polar DNA bases and when the energy needed to polarize the electron den- sity of the solute has been considered in the transfer free energy. These results were further confirmed by hydration free energies of polar and aromatic amino acid side chain analogues. Comparison of the two partitioning methods Hirsheld-I and Minimal Basis Iterative Stockholder (MBIS) revealed some deficiencies in the Hirshfeld-I method related to nonexistent isolated anionic nitrogen pro-atoms used in the method. Hydration free energies and partitioning coefficients obtained with atomic charges from the MBIS partitioning method accounting for polarization by the implicit solvation model are in good agreement with the experimental values. </p> </div> </div> </div>

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Faculty Opinions recommendation of Quantitative interaction proteomics and genome-wide profiling of epigenetic histone marks and their readers.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.5781959.5764057 ◽

2010 ◽

Author(s):

Peter Park

Keyword(s):

Histone Marks ◽

Genome Wide ◽

Interaction Proteomics

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Cardiomyocyte-Specific Circulating Cell-Free Methylated DNA in Esophageal Cancer Patients Treated with Chemoradiation

Gastrointestinal Disorders ◽

10.3390/gidisord3030011 ◽

2021 ◽

Vol 3 (3) ◽

pp. 100-112

Author(s):

Sarah Martinez Roth ◽

Eveline E. Vietsch ◽

Megan E. Barefoot ◽

Marcel O. Schmidt ◽

Matthew D. Park ◽

...

Keyword(s):

Esophageal Cancer ◽

Cardiac Toxicity ◽

Amplicon Sequencing ◽

Neoadjuvant Chemoradiation ◽

Patient Specific ◽

High Dose ◽

Specific Cell ◽

Methylated Dna ◽

Bisulfite Treatment ◽

Dose Radiation

Thoracic high-dose radiation therapy (RT) for cancer has been associated with early and late cardiac toxicity. To assess altered rates of cardiomyocyte cell death due to RT we monitored changes in cardiomyocyte-specific, cell-free methylated DNA (cfDNA) shed into the circulation. Eleven patients with distal esophageal cancer treated with neoadjuvant chemoradiation to 50.4 Gy (RT) and concurrent carboplatin and paclitaxel were enrolled. Subjects underwent fasting blood draws prior to the initiation and after completion of RT as well as 4–6 months following RT. An island of six unmethylated CpGs in the FAM101A locus was used to identify cardiomyocyte-specific cfDNA in serum. After bisulfite treatment this specific cfDNA was quantified by amplicon sequencing at a depth of >35,000 reads/molecule. Cardiomyocyte-specific cfDNA was detectable before RT in the majority of patient samples and showed some distinct changes during the course of treatment and recovery. We propose that patient-specific cardiac damages in response to the treatment are indicated by these changes although co-morbidities may obscure treatment-specific events.

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Structural insights into the repair mechanism of AGT for methyl-induced DNA damage

Biological Chemistry ◽

10.1515/hsz-2021-0198 ◽

2021 ◽

Vol 0 (0) ◽

Author(s):

Rajendra P. Koirala ◽

Rudramani Pokhrel ◽

Prabin Baral ◽

Purushottam B. Tiwari ◽

Prem P. Chapagain ◽

...

Keyword(s):

Covalent Bond ◽

Cancer Therapeutics ◽

Md Simulations ◽

Structural Features ◽

Umbrella Sampling ◽

Repair Mechanism ◽

Structural Investigations ◽

Methylated Dna ◽

Dna Base ◽

Dna Alkyltransferase

Abstract Methylation induced DNA base-pairing damage is one of the major causes of cancer. O6-alkylguanine-DNA alkyltransferase (AGT) is considered a demethylation agent of the methylated DNA. Structural investigations with thermodynamic properties of the AGT-DNA complex are still lacking. In this report, we modeled two catalytic states of AGT-DNA interactions and an AGT-DNA covalent complex and explored structural features using molecular dynamics (MD) simulations. We utilized the umbrella sampling method to investigate the changes in the free energy of the interactions in two different AGT-DNA catalytic states, one with methylated GUA in DNA and the other with methylated CYS145 in AGT. These non-covalent complexes represent the pre- and post-repair complexes. Therefore, our study encompasses the process of recognition, complex formation, and separation of the AGT and the damaged (methylated) DNA base. We believe that the use of parameters for the amino acid and nucleotide modifications and for the protein-DNA covalent bond will allow investigations of the DNA repair mechanism as well as the exploration of cancer therapeutics targeting the AGT-DNA complexes at various functional states as well as explorations via stabilization of the complex.

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Isolation of DNA fragments associated with methylated CpG islands in human adenocarcinomas of the lung using a methylated DNA binding column and denaturing gradient gel electrophoresis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.96.6.2913 ◽

1999 ◽

Vol 96 (6) ◽

pp. 2913-2918 ◽

Cited By ~ 29

Author(s):

M. Shiraishi ◽

Y. H. Chuu ◽

T. Sekiya

Keyword(s):

Dna Binding ◽

Gel Electrophoresis ◽

Denaturing Gradient Gel Electrophoresis ◽

Cpg Islands ◽

Dna Fragments ◽

Methylated Dna ◽

Gradient Gel Electrophoresis

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The major histocompatibility complex class II promoter-binding protein RFX (NF-X) is a methylated DNA-binding protein.

Molecular and Cellular Biology ◽

10.1128/mcb.13.11.6810 ◽

1993 ◽

Vol 13 (11) ◽

pp. 6810-6818 ◽

Cited By ~ 33

Author(s):

X Y Zhang ◽

N Jabrane-Ferrat ◽

C K Asiedu ◽

S Samac ◽

B M Peterlin ◽

...

Keyword(s):

Dna Methylation ◽

Dna Binding ◽

Dna Sequences ◽

Protein Complexes ◽

Binding Protein ◽

Dna Binding Protein ◽

Class Ii ◽

Major Histocompatibility ◽

Methylated Dna ◽

Mammalian Protein

A mammalian protein called RFX or NF-X binds to the X box (or X1 box) in the promoters of a number of major histocompatibility (MHC) class II genes. In this study, RFX was shown to have the same DNA-binding specificity as methylated DNA-binding protein (MDBP), and its own cDNA was found to contain a binding site for MDBP in the leader region. MDBP is a ubiquitous mammalian protein that binds to certain DNA sequences preferentially when they are CpG methylated and to other related sequences, like the X box, irrespective of DNA methylation. MDBP from HeLa and Raji cells formed DNA-protein complexes with X-box oligonucleotides that coelectrophoresed with those containing standard MDBP sites. Furthermore, MDBP and X-box oligonucleotides cross-competed for the formation of these DNA-protein complexes. DNA-protein complexes obtained with MDBP sites displayed the same partial supershifting with an antiserum directed to the N terminus of RFX seen for complexes containing an X-box oligonucleotide. Also, the in vitro-transcribed-translated product of a recombinant RFX cDNA bound specifically to MDBP ligands and displayed the DNA methylation-dependent binding of MDBP. RFX therefore contains MDBP activity and thereby also EF-C, EP, and MIF activities that are indistinguishable from MDBP and that bind to methylation-independent sites in the transcriptional enhancers of polyomavirus and hepatitis B virus and to an intron of c-myc.

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