5-Azacytidine induced methyltransferase-DNA adducts block DNA replication in vivo
| Autor: | Kenneth N. Kreuzer, Jack D. Griffith, H. Kenny Kuo |
|---|---|
| Rok vydání: | 2007 |
| Předmět: |
DNA Replication
Cancer Research DNA-Cytosine Methylases Point mutation DNA replication Cytidine Biology Molecular biology Models Biological chemistry.chemical_compound DNA Adducts Rec A Recombinases Replication factor C Oncology chemistry Control of chromosome duplication DNA methylation Azacitidine Escherichia coli DNA S phase Plasmids |
| Zdroj: | Cancer research. 67(17) |
| ISSN: | 0008-5472 |
| Popis: | 5-Azacytidine (aza-C) and its derivatives are cytidine analogues used for leukemia chemotherapy. The primary effect of aza-C is the prohibition of cytosine methylation, which results in covalent methyltransferase-DNA (MTase-DNA) adducts at cytosine methylation sites. These adducts have been suggested to cause chromosomal rearrangements and contribute to cytotoxicity, but the detailed mechanisms have not been elucidated. We used two-dimensional agarose gel electrophoresis and electron microscopy to analyze plasmid pBR322 replication dynamics in Escherichia coli cells grown in the presence of aza-C. Two-dimensional gel analysis revealed the accumulation of specific bubble and Y molecules, dependent on overproduction of the cytosine MTase EcoRII (M.EcoRII) and treatment with aza-C. Furthermore, a point mutation that eliminates a particular EcoRII methylation site resulted in disappearance of the corresponding bubble and Y molecules. These results imply that aza-C–induced MTase-DNA adducts block DNA replication in vivo. RecA-dependent X structures were also observed after aza-C treatment. These molecules may be generated from blocked forks by recombinational repair and/or replication fork regression. In addition, electron microscopy analysis revealed both bubbles and rolling circles (RC) after aza-C treatment. These results suggest that replication can switch from theta to RC mode after a replication fork is stalled by an MTase-DNA adduct. The simplest model for the conversion of theta to RC mode is that the blocked replication fork is cleaved by a branch-specific endonuclease. Such replication-dependent DNA breaks may represent an important pathway that contributes to genome rearrangement and/or cytotoxicity. [Cancer Res 2007;67(17):8248–54] |
| Databáze: | OpenAIRE |
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