5-Azacytidine–Induced Methyltransferase-DNA Adducts Block DNA Replication In vivo

H Kenny Kuo,Kenneth N Kreuzer,Jack D Griffith

doi:10.1158/0008-5472.can-07-1038

Abstract

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.

Highlights

Numerous chemicals as well as radiation can lead to covalent protein-DNA adducts, but little is known about the consequences of these adducts in vivo
We first analyzed DNA isolated from a pBR322-containing strain that has only the endogenous DNA cytosine MTase (Dcm)
The activity of endogenous Dcm protein is weak [26]; we reasoned that MTase-DNA adducts formed with Dcm might be insufficient for detecting stalled forks

Summary

Introduction

Numerous chemicals as well as radiation can lead to covalent protein-DNA adducts, but little is known about the consequences of these adducts in vivo. One important chemotherapeutic agent that leads to protein-DNA adducts is 5-azacytidine (aza-C), which was first synthesized in 1964 [1]. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Aza-C substitution at the target cytosine interferes with the reaction cycle and results in long-lived or irreversible MTase-DNA adducts [6,7,8]. Evidence for adduct formation in vivo has been presented [9, 10]

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Conclusion