title: A polymerization-based method to construct a plasmid containing clustered DNA damage and a mismatch.
abstract: Exposure of biological materials to ionizing radiation often induces clustered DNA damage. The mutagenicity of clustered DNA damage can be analyzed with plasmids carrying a clustered DNA damage site, in which the strand bias of a replicating plasmid (i.e., the degree to which each of the two strands of the plasmid are used as the template for replication of the plasmid) can help to clarify how clustered DNA damage enhances the mutagenic potential of comprising lesions. Placement of a mismatch near a clustered DNA damage site can help to determine the strand bias, but present plasmid-based methods do not allow insertion of a mismatch at a given site in the plasmid. Here, we describe a polymerization-based method for constructing a plasmid containing clustered DNA lesions and a mismatch. The presence of a DNA lesion and a mismatch in the plasmid was verified by enzymatic treatment and by determining the relative abundance of the progeny plasmids derived from each of the two strands of the plasmid.
authors.affiliation: Quantum Beam Science Research Directorate, National Institutes of Quantum and Radiological Science and Technology, Japan.
authors.forename: Momoko
authors.initials: M
authors.lastname: Takahashi
authors.affiliation: Quantum Beam Science Research Directorate, National Institutes of Quantum and Radiological Science and Technology, Japan.
authors.forename: Ken
authors.initials: K
authors.lastname: Akamatsu
authors.affiliation: Quantum Beam Science Research Directorate, National Institutes of Quantum and Radiological Science and Technology, Japan. Electronic address: shikazono.naoya@qst.go.jp.
authors.forename: Naoya
authors.initials: N
authors.lastname: Shikazono
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D004249:DNA Damage; D004926:Escherichia coli / Q000235:genetics*; D010957:Plasmids / Q000235:genetics*
keywords: Clustered DNA damage; Mismatch; Mutagenesis