title: Insights into mutagenesis using Escherichia coli chromosomal lacZ strains that enable detection of a wide spectrum of mutational events.
abstract: Strand misalignments at DNA repeats during replication are implicated in mutational hotspots. To study these events, we have generated strains carrying mutations in the Escherichia coli chromosomal lacZ gene that revert via deletion of a short duplicated sequence or by template switching within imperfect inverted repeat (quasipalindrome, QP) sequences. Using these strains, we demonstrate that mutation of the distal repeat of a quasipalindrome, with respect to replication fork movement, is about 10-fold higher than the proximal repeat, consistent with more common template switching on the leading strand. The leading strand bias was lost in the absence of exonucleases I and VII, suggesting that it results from more efficient suppression of template switching by 3' exonucleases targeted to the lagging strand. The loss of 3' exonucleases has no effect on strand misalignment at direct repeats to produce deletion. To compare these events to other mutations, we have reengineered reporters (designed by Cupples and Miller 1989) that detect specific base substitutions or frameshifts in lacZ with the reverting lacZ locus on the chromosome rather than an F' element. This set allows rapid screening of potential mutagens, environmental conditions, or genetic loci for effects on a broad set of mutational events. We found that hydroxyurea (HU), which depletes dNTP pools, slightly elevated templated mutations at inverted repeats but had no effect on deletions, simple frameshifts, or base substitutions. Mutations in nucleotide diphosphate kinase, ndk, significantly elevated simple mutations but had little effect on the templated class. Zebularine, a cytosine analog, elevated all classes.
authors: Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02454, USA.
authors: Tracey
authors: T
authors: Seier
authors: Dana R
authors: DR
authors: Padgett
authors: Gal
authors: G
authors: Zilberberg
authors: Vincent A
authors: VA
authors: Sutera
authors: Noor
authors: N
authors: Toha
authors: Susan T
authors: ST
authors: Lovett
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002876:Chromosomes, Bacterial / Q000235:genetics; D003562:Cytidine / Q000031:analogs & derivatives / Q000494:pharmacology; D004252:DNA Mutational Analysis / Q000379:methods*; D004261:DNA Replication / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics*; D016368:Frameshift Mutation; D006918:Hydroxyurea / Q000494:pharmacology; D055029:Inverted Repeat Sequences / Q000235:genetics; D007763:Lac Operon / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation / Q000187:drug effects; D009690:Nucleic Acid Conformation; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D017384:Sequence Deletion
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D019384:Nucleic Acid Synthesis Inhibitors; D003562:Cytidine; C009131:pyrimidin-2-one beta-ribofuranoside; D006918:Hydroxyurea