Preferential DNA secondary structure mutagenesis in the lagging strand of replication in E. coli.
When present in single-stranded DNA, palindromic or quasi-palindromic sequences have the potential to form complex secondary structures, including hairpins, which may facilitate interstrand misalignment of direct repeats and be responsible for diverse types of replication-based mutations, including deletions, additions, frameshifts and duplications. In regions of palindromic symmetry, specific deletion events may involve the formation of a hairpin or other DNA secondary structures which can stabilize the misalignment of direct repeats. One model suggests that these deletions occur during DNA replication by slippage of the template strand and misalignment with the progeny strand. The concurrent DNA replication model, involving an asymmetric dimeric DNA polymerase III complex which replicates the leading and lagging strands, has significant implications for mutagenesis. The intermittent looping of the lagging strand template, and the fact that the lagging strand template may contain a region of single-stranded DNA the length of an Okazaki fragment, provides an opportunity for DNA secondary-structure formation and misalignment. Here we report our design of a palindromic fragment to create an 'asymmetric palindromic insert' in the chloramphenicol acetyltransferase gene of plasmid pBR325. The frequency with which the insert was deleted in Escherichia coli depends on the orientation of the gene in the plasmid. Our results suggest that replication-dependent deletion between direct repeats may occur preferentially in the lagging strand.
Trinh
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TQ
Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Ohio 45267-0524.
Sinden
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RR
D001483:Base Sequence; D002872:Chromosome Deletion; D004261:DNA Replication; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009690:Nucleic Acid Conformation; D010957:Plasmids
D004269:DNA, Bacterial