Dataset Information

Thermodynamic and kinetic basis for recognition and repair of 8-oxoguanine in DNA by human 8-oxoguanine-DNA glycosylase.

ABSTRACT: We have used a stepwise increase in ligand complexity approach to estimate the relative contributions of the nucleotide units of DNA containing 7,8-dihydro-8-oxoguanine (oxoG) to its total affinity for human 8-oxoguanine DNA glycosylase (OGG1) and construct thermodynamic models of the enzyme interaction with cognate and non-cognate DNA. Non-specific OGG1 interactions with 10-13?nt pairs within its DNA-binding cleft provides approximately 5 orders of magnitude of its affinity for DNA (?G°?approximately -6.7?kcal/mol). The relative contribution of the oxoG unit of DNA (?G° approximately -3.3?kcal/mol) together with other specific interactions (?G° approximately -0.7?kcal/mol) provide approximately 3 orders of magnitude of the affinity. Formation of the Michaelis complex of OGG1 with the cognate DNA cannot account for the major part of the enzyme specificity, which lies in the k(cat) term instead; the rate increases by 6-7 orders of magnitude for cognate DNA as compared with non-cognate one. The k(cat) values for substrates of different sequences correlate with the DNA twist, while the K(M) values correlate with ?G° of the DNA fragments surrounding the lesion (position from -6 to +6). The functions for predicting the K(M) and k(cat) values for different sequences containing oxoG were found.

SUBMITTER: Kirpota OO

PROVIDER: S-EPMC3113562 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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Thermodynamic and kinetic basis for recognition and repair of 8-oxoguanine in DNA by human 8-oxoguanine-DNA glycosylase.

Kirpota Oleg O OO Endutkin Anton V AV Ponomarenko Michail P MP Ponomarenko Petr M PM Zharkov Dmitry O DO Nevinsky Georgy A GA

Nucleic acids research 20110222 11

We have used a stepwise increase in ligand complexity approach to estimate the relative contributions of the nucleotide units of DNA containing 7,8-dihydro-8-oxoguanine (oxoG) to its total affinity for human 8-oxoguanine DNA glycosylase (OGG1) and construct thermodynamic models of the enzyme interaction with cognate and non-cognate DNA. Non-specific OGG1 interactions with 10-13 nt pairs within its DNA-binding cleft provides approximately 5 orders of magnitude of its affinity for DNA (ΔG° approxi ...[more]

PMID: 21343179

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Project description:Purpose: The aim of this study is to test whether global gene expression induced by multiple challenges with OGG1-BER product 8-oxoG in mouse airways is linked to airway remodeling. Methods: RNAs extracted from individual mouse lungs (experimental group: n=5) were pooled and a total 1 ?g RNA was used for Next-Generation Sequencing (NGS) analyses on an Illumina HiSeq 1000 sequencing system. Sequence analyses were performed in duplicate. First- and second-strand synthesis, adapter ligation and amplification of the library were performed using the Illumina TruSeq Sample Preparation Kit as recommended by the manufacturer (Illumina). Library quailty was evaluated by using an Agilent DNA-1000 chip on an Agilent 2100 Bioanalyzer. Library DNA templates were quantitated by qPCR using known reference standards. Cluster formation of the library of DNA templates was performed using the TruSeq PE Cluster Kit v3 (Illumina) and the Illumina cBot workstation. Paired-end, 50-base sequencing was performed with a TruSeq SBS kit v3 (Illumina) on the Illumina HiSeq 1000 by protocols defined by the manufacturer. Base call conversion to sequence reads was performed using CASAVA-1.8.2. Sequence data were analyzed with the Bowtie2, Tophat2 and GFOLD programs. Processed data are presented as reads per kilobase transcript per million (RPKM), normalized to the experimental control (RNA from saline challenged lungs) and reported as fold change (test/control). Results: We mapped an average of 31.41 million sequence reads per sample and identified 23,337 transcripts in total RNA extracted from lungs of Balb/cJ mice as described in Methods. Approximately 14% of the transcripts showed differential expression between the saline-challenged control and 8-oxoguanine-challeged mouse lungs, with a fold change ?3.0. We validated the expression changes of 18 selected EMT-related genes of interest for our studies by qRT-PCR. Hierarchical clustering followed by Protein ANalysis THrough Evolutionary Relationships database (PANTHER) analysis of differentially expressed genes was done using GENE-E online software from Broad Institute (http://www.broadinstitute.org/cancer/software/GENE-E/). Results from PANTHER analysis of upregulated transcripts (fold change ?3.0) showed overrepresentation of various biological functions (GO terms) including developmental process (GO:0032502, P=4.58E-33), system development (GO:0048731, P=9.16E-33), cellular process (GO:0009987, P= 5.52E-31), cell adhesion (GO:0007155, P= 8.63E-28) among others. Pathway analysis (PANTHER) indicated that the most overrepresented pathways were: cadherin signaling (P00012, P=6.62E-07), wnt signaling (P00057, P= 5.81E-06), integrin signaling (P00034, P= 1.09E-05) among others. In addition to gene expression analysis, we confirmed airway remodeling by histological examination (Hematoxylin and Eosin, Masson's trichrome staining) of lung sections at seven days from the last challenge (day 11). Conclusions: This is the first study showing a link between gene expression at whole-transcriptome level induced by chronic OGG1-BER (mimicked by multiple challenges with 8-oxoG) and airway remodeling, supported by histological structural changes in lungs. Balb/cJ mice (5 per group) were intranasally challenged with 8-oxoguanine (1 µM, 60 µl) for three times at days 0, 2 and 4. Control group mice were intranasally challenged with saline (60 µl). At 30, 60 and 120 min after the third challenge (day 4), mice were sacrificed and lungs were processed for RNA extraction. RNAs from individual mice within the same group were pooled and subjected to deep-sequencing analysis in duplicate using NSG on an Illumina HiSeq 1000 sequencing system. After alignment and processing, the resulting RPKM from treatment groups (8-oxoG-challenged) were normalized to control group (saline-challenged).

Dataset Information

Thermodynamic and kinetic basis for recognition and repair of 8-oxoguanine in DNA by human 8-oxoguanine-DNA glycosylase.

Publications

Thermodynamic and kinetic basis for recognition and repair of 8-oxoguanine in DNA by human 8-oxoguanine-DNA glycosylase.

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