Understanding the structural and dynamic consequences of DNA epigenetic modifications: Computational insights into cytosine methylation and hydroxymethylation

We report a series of molecular dynamics (MD) simulations of up to a microsecond combined simulation time designed to probe epigenetically modified DNA sequences. More specifically, by monitoring the effects of methylation and hydroxymethylation of cytosine in different DNA sequences, we show, for the first time, that DNA epigenetic modifications change the molecule's dynamical landscape, increasing the propensity of DNA toward different values of twist and/or roll/tilt angles (in relation to the unmodified DNA) at the modification sites. Moreover, both the extent and position of different modifications have significant effects on the amount of structural variation observed. We propose that these conformational differences, which are dependent on the sequence environment, can provide specificity for protein binding.     

Detecting DNA Modifications from SMRT Sequencing Data by Modeling Sequence Context Dependence of Polymerase Kinetic

DNA modifications such as methylation and DNA damage can play critical regulatory roles in biological systems. Single molecule, real time (SMRT) sequencing technology generates DNA sequences as well as DNA polymerase kinetic information that can be used for the direct detection of DNA modifications. We demonstrate that local sequence context has a strong impact on DNA polymerase kinetics in the neighborhood of the incorporation site during the DNA synthesis reaction, allowing for the possibility of estimating the expected kinetic rate of the enzyme at the incorporation site using kinetic rate information collected from existing SMRT sequencing data (historical data) covering the same local sequence contexts of interest. We develop an Empirical Bayesian hierarchical model for incorporating historical data. Our results show that the model could greatly increase DNA modification detection accuracy, and reduce requirement of control data coverage. For some DNA modifications that have a strong signal, a control sample is not even needed by using historical data as alternative to control. Thus, sequencing costs can be greatly reduced by using the model. We implemented the model in a R package named seqPatch, which is available at https://github.com/zhixingfeng/seqPatch.     

Using Triplet Periodicity of Nucleotide Sequences for Finding Potential Reading Frame Shifts in Genes

We introduce a novel approach for the detection of possible mutations leading to a reading frame (RF) shift in a gene. Deletions and insertions of DNA coding regions are considerable events for genes because an RF shift results in modifications of the extensive region of amino acid sequence coded by a gene. The suggested method is based on the phenomenon of triplet periodicity (TP) in coding regions of genes and its relative resistance to substitutions in DNA sequence. We attempted to extend 326 933 regions of continuous TP found in genes from the KEGG databank by considering possible insertions and deletions. We revealed totally 824 genes where such extension was possible and statistically significant. Then we generated amino acid sequences according to active (KEGG''s) and hypothetically ancient RFs in order to find confirmation of a shift at a protein level. Consequently, 64 sequences have protein similarities only for ancient RF, 176 only for active RF, 3 for both and 581 have no protein similarity at all. We aimed to have revealed lower bound for the number of genes in which a shift between RF and TP is possible. Further ways to increase the number of revealed RF shifts are discussed.     

A quantitative model of nucleosome dynamics

The expression, replication and repair of eukaryotic genomes require the fundamental organizing unit of chromatin, the nucleosome, to be unwrapped and disassembled. We have developed a quantitative model of nucleosome dynamics which provides a fundamental understanding of these DNA processes. We calibrated this model using results from high precision single molecule nucleosome unzipping experiments, and then tested its predictions for experiments in which nucleosomes are disassembled by the DNA mismatch recognition complex hMSH2-hMSH6. We found that this calibrated model quantitatively describes hMSH2-hMSH6 induced disassembly rates of nucleosomes with two separate DNA sequences and four distinct histone modification states. In addition, this model provides mechanistic insight into nucleosome disassembly by hMSH2-hMSH6 and the influence of histone modifications on this disassembly reaction. This model''s precise agreement with current experiments suggests that it can be applied more generally to provide important mechanistic understanding of the numerous nucleosome alterations that occur during DNA processing.     

DNA conformation and dynamics

Nucleotide conformation and dynamics are important for the study of radiation damage to DNA at the atomic level. It is necessary to study not only normal oligonucleotide structure but also those containing modified bases which result from interaction with OH-radicals. There are now over 8000 atomic coordinate entries in the Brookhaven Protein Data Bank, of which over 900 relate to experimentally determined structures of nucleic acids and nucleic acid/protein complexes. We review some of these data which have led to the elucidation of novel DNA conformations, insight into DNA sequence specificity and knowledge of protein/DNA interactions. Further understanding of the conformation, stability and dynamics of nucleic acids has come from molecular modelling. We have used such techniques to study chemical modifications to bases such as alkylation of thymine and guanine and the effects of curvature in longer sequences. Recent improvements in this area include the inclusions of explicit counter-ions and solvent molecules, the use of Particle Mesh Ewald methods to incorporate the long-range electrostatic interactions and the use of longer time scale simulations. We have employed these methods to analyse the effects of incorporation of 8-oxodeoxyguanosine into duplex DNA. This lesion is a common result of radiation damage and is known to have important effects in mutagenesis, cancer and ageing. Received: 7 October 1998 / Accepted in revised form: 18 January 1999     

Effects of excess centromeres and excess telomeres on chromosome loss rates.

The linear chromosomes of eukaryotes contain specialized structures to ensure their faithful replication and segregation to daughter cells. Two of these structures, centromeres and telomeres, are limited, respectively, to one and two copies per chromosome. It is possible that the proteins that interact with centromere and telomere DNA sequences are present in limiting amounts and could be competed away from the chromosomal copies of these elements by additional copies introduced on plasmids. We have introduced excess centromeres and telomeres into Saccharomyces cerevisiae and quantitated their effects on the rates of loss of chromosome III and chromosome VII by fluctuation analysis. We show that (i) 600 new telomeres have no effect on chromosome loss; (ii) an average of 25 extra centromere DNA sequences increase the rate of chromosome III loss from 0.4 x 10(-4) events per cell division to 1.3 x 10(-3) events per cell division; (iii) centromere DNA (CEN) sequences on circular vectors destabilize chromosomes more effectively than do CEN sequences on 15-kb linear vectors, and transcribed CEN sequences have no effect on chromosome stability. We discuss the different effects of extra centromere and telomere DNA sequences on chromosome stability in terms of how the cell recognizes these two chromosomal structures.     

APE1 Incision Activity at Abasic Sites in Tandem Repeat Sequences

Repetitive DNA sequences, such as those present in microsatellites and minisatellites, telomeres, and trinucleotide repeats (linked to fragile X syndrome, Huntington disease, etc.), account for nearly 30% of the human genome. These domains exhibit enhanced susceptibility to oxidative attack to yield base modifications, strand breaks, and abasic sites; have a propensity to adopt non-canonical DNA forms modulated by the positions of the lesions; and, when not properly processed, can contribute to genome instability that underlies aging and disease development. Knowledge on the repair efficiencies of DNA damage within such repetitive sequences is therefore crucial for understanding the impact of such domains on genomic integrity. In the present study, using strategically designed oligonucleotide substrates, we determined the ability of human apurinic/apyrimidinic endonuclease 1 (APE1) to cleave at apurinic/apyrimidinic (AP) sites in a collection of tandem DNA repeat landscapes involving telomeric and CAG/CTG repeat sequences. Our studies reveal the differential influence of domain sequence, conformation, and AP site location/relative positioning on the efficiency of APE1 binding and strand incision. Intriguingly, our data demonstrate that APE1 endonuclease efficiency correlates with the thermodynamic stability of the DNA substrate. We discuss how these results have both predictive and mechanistic consequences for understanding the success and failure of repair protein activity associated with such oxidatively sensitive, conformationally plastic/dynamic repetitive DNA domains.     

单分子实时测序及其在微生物表观遗传学中的应用

表观遗传学对于微生物的生命进程起着重要作用。由限制-修饰系统调控的DNA修饰参与微生物的免疫防御系统,无限制-修饰系统调控的DNA修饰通过调控基因表达影响表型。然而,表观遗传信息还没有被常规地作为DNA信息收集分析。基于对DNA合成反应的动力学分析,单分子实时测序技术可以在获得基本序列数据的同时实现对被修饰核苷酸的检测。这个技术为微生物中已知DNA修饰的研究提供了新的平台,也为新型DNA修饰的发现做好准备。本文综述了单分子实时测序技术及其在微生物表观遗传学中的应用。     

A systematic molecular dynamics study of nearest-neighbor effects on base pair and base pair step conformations and fluctuations in B-DNA

It is well recognized that base sequence exerts a significant influence on the properties of DNA and plays a significant role in protein–DNA interactions vital for cellular processes. Understanding and predicting base sequence effects requires an extensive structural and dynamic dataset which is currently unavailable from experiment. A consortium of laboratories was consequently formed to obtain this information using molecular simulations. This article describes results providing information not only on all 10 unique base pair steps, but also on all possible nearest-neighbor effects on these steps. These results are derived from simulations of 50–100 ns on 39 different DNA oligomers in explicit solvent and using a physiological salt concentration. We demonstrate that the simulations are converged in terms of helical and backbone parameters. The results show that nearest-neighbor effects on base pair steps are very significant, implying that dinucleotide models are insufficient for predicting sequence-dependent behavior. Flanking base sequences can notably lead to base pair step parameters in dynamic equilibrium between two conformational sub-states. Although this study only provides limited data on next-nearest-neighbor effects, we suggest that such effects should be analyzed before attempting to predict the sequence-dependent behavior of DNA.     

DNA methylase and demethylase activities are modulated by one-carbon metabolism in Alzheimer's disease models

Late-onset Alzheimer's disease seems to be a multi-factorial disease with both genetic and non-genetic, environmental, possible causes. Recently, epigenomics is achieving a major role in Alzheimer's research due to its involvement in different molecular pathways leading to neurodegeneration. Among the different epigenetic modifications, DNA methylation is one of the most relevant to the disease. We previously demonstrated that presenilin1 (PSEN1), a gene involved in amyloidogenesis, is modulated by DNA methylation in neuroblastoma cells and Alzheimer's mice in an experimental model of nutritionally altered one-carbon metabolism. This alteration, obtained by nutritional deficiency of B vitamins (folate, B12 and B6) hampered S-adenosylmethionine (SAM)-dependent methylation reactions. The aim of the present paper was to investigate the regulation of DNA methylation machinery in response to hypomethylating (B vitamin deficiency) and hypermethylating (SAM supplementation) alterations of the one-carbon metabolism. We found that DNA methylases (DNMT1, 3a and 3b) and a putative demethylase (MBD2) were differently modulated, in line with the previously observed changes of PSEN1 methylation pattern in the same experimental conditions.