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

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 branchspecific 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]

Error-prone Translesion Synthesis Past DNA-Peptide Cross-links Conjugated to the Major Groove of DNA via C5 of Thymidine

“…The modified nucleotide was added using an off-line manual coupling protocol. A biotinylated 23-mer primer (biotin-5Ј-(T) 10 GGG GGA AGG AUT C-3Ј) and 13-mer primer (5Ј-GGG GGA AGG ATT C-3Ј) were purchased from Integrated DNA Technologies (Coralville, IA). All oligodeoxynucleotides were purified by semi-preparative HPLC, desalted by Illustra NAP-5 columns, characterized by HPLC-ESI Ϫ -MS, and quantified by UV spectrophotometry as described previously (37).…”

“…Primer-template duplexes used for HPLC-ESI Ϫ -MS/MS sequencing experiments were generated analogously by annealing biotinylated 23-mer primer (biotin-5Ј-(T) 10 GGG GGA AGG AUT C-3Ј, 100 pmol) to template strands (5Ј-TCA TXG AAT CCT TCC CCC-3Ј) containing either unmodified dT or the dT-peptide conjugate at position X (2 eq. ).…”

“…Because of their bulky nature, DPCs can block human replicative DNA polymerases ␦ and ⑀, stalling cellular replication (10). A specialized group of translesion synthesis (TLS) polymerases (human Y family polymerases , , , and Rev1, B family polymerase , and human A family polymerase ) can be recruited to blocked replication forks (11)(12)(13)(14)(15)(16)(17)(18).…”

“…Previous investigations have revealed that the ability of DNA polymerases to bypass DNA-peptide conjugates is dependent on the lesion size, the attachment site within the DNA, and polymerase identity (10,(21)(22)(23)(24)(25)(26)(27)(28)(29). Lloyd and co-workers (26) have reported that human polymerase (hpol) and its Escherichia coli orthologue pol IV were able to catalyze error-free primer extension past DNA templates containing tetra-and dodecapeptides conjugated to the N 2 position of guanine via a trimethylene linker.…”

Modulation of UvrD Helicase Activity by Covalent DNA-Protein Cross-links

“…For example, treatment with a cytidine analog, azacytidine C, results in covalently linked methyltransferase-DNA adducts that cause blockage of DNA replication in vivo (49). This blockage may be overcome by the action of specialized, translesion synthesis DNA polymerases that have been shown to be required for bypassing DNA-peptide cross-links, the potential intermediates generated during processing of DPCs (50).…”