Asymmetric cytosine deamination revealed by spontaneous mutational specificity in an Ung− strain of Escherichia coli

Abstract

A collection of 164 spontaneous lacI- mutations were recovered from a uracil-DNA glycosylase deficient (Ung-) strain of Escherichia coli and analyzed by DNA sequencing. As predicted by genetic studies, G:C----A:T transitions predominated among base substitution events. However, DNA sequence analysis indicated that these events did not occur at random. Of the 31 G:C----A:T transitions recovered, 24 involved cytosine residues located in the nontranscribed strand of the gene and 15 of the 31 transitions occurred at cytosines located on the 3' side of 3 or more A:T base pairs. These differentials likely reflect the more single-stranded character of the non-transcribed strand of the gene and of regions rich in A:T base pairs. In addition, mutation at the frameshift hotspot was altered in the Ung- strain, suggesting a role for DNA repair in the formation of structural intermediates that potentiate these events. Also, the analysis of non-hotspot frameshifts, deletions and duplications showed that many involved local DNA sequence. Specifically, several of the frameshift, deletion and duplication mutations occurred near the sequence 5'-CTGG-3'. Thus, DNA sequence analysis of mutational specificity in an Ung- strain has provided evidence that gene expression, DNA repair and DNA context can all potentially influence the classes and frequencies of spontaneous mutation.

Spectrum of spontaneous mutation at the APRT locus of Chinese hamster ovary cells: an analysis at the DNA sequence level.

“…Eighteen of the 22 independent mutants of this type (or 9 of 12 individual sites of GC to APT mutations) were read as cytosine to thymine changes in the single-stranded DNA used for dideoxy sequencing, which is, in this case, the nontranscribed strand. Such a preference was also observed in an Ung-strain of E. coli (34). The predominance of mutation in the nontranscribed strand in E. coli was thought to reflect the single-stranded nature of this strand during transcription, while the transcribed strand is complexed with RNA.…”

A Biochemical Mechanism for Nonrandom Mutations and Evolution

“…This process occurs 140 times more frequently in single-stranded DNA (ssDNA) than in double-stranded DNA (dsDNA) (in which N-3 is hydrogen bonded to N-1 of guanine), in mismatched base pairs, and in AT-rich regions that "breathe." These background mutations and frameshift-induced deletions and additions are DNA sequence directed in that they occur most frequently in ssDNA and in unpaired, mispaired, and methylated bases (15,26,28,31,32,60). Such vulnerable bases can arise as a consequence of slippage in tandem repeats or as a result of stem-loop DNA secondary structures that arise from sequences containing intrastrand inverted complements.…”

“…The most common base substitution events in the spectra of background mutations in E. coli and mammalian cells are G ⅐ C-to-A ⅐ T transitions. Fix and Glickman (28) observe that 77% of these mutations originate on the nontranscribed strand in E. coli mutants unable to repair deaminated cytosines. This suggests that the unprotected single strand in the transcription "bubble" is significantly more vulnerable to mutations than the transcribed strand, which is protected as a DNA-RNA hybrid (Fig.…”

Sequence specificity for removal of uracil from U·A pairs and U·G mismatches by uracil‐DNA glycosylase from Escherichia coli, and correlation with mutational hotspots

“…While many of these mutations may be caused by deamination of 5-methylcytosine to thymine, deamination of cytosine to uracil may also contribute significantly [6][7][8][9][10]. Uracil-DNA glycosylase removes uracil from DNA, thus initiating the base excisison repair pathway for removal of uracil in DNA [11].…”