Transcription-Dependent Increase in Multiple Classes of Base Substitution Mutations in
            <i>Escherichia coli</i>

Klapacz, Joanna; Bhagwat, Ashok S.

doi:10.1128/jb.184.24.6866-6872.2002

Cited by 39 publications

(39 citation statements)

References 21 publications

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“…None of these values is significantly different from expected (P ≥ 0.42 in every case). Thus, our results do not support previous observations that highly expressed genes have either high (11,35,36) or low (37-39) mutation rates. Unlike a previous report (39), we found no bias for any type of base change occurring preferentially on either the transcribed or the nontranscribed DNA strand (Table S3).…”

Section: Distribution Of Mutations Is Random Among Wild-type Lines Bucontrasting

confidence: 99%

Rate and molecular spectrum of spontaneous mutations in the bacteriumEscherichia colias determined by whole-genome sequencing

Lee¹,

Popodi

Tang³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

648

131

859

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Knowledge of the rate and nature of spontaneous mutation is fundamental to understanding evolutionary and molecular processes. In this report, we analyze spontaneous mutations accumulated over thousands of generations by wild-type Escherichia coli and a derivative defective in mismatch repair (MMR), the primary pathway for correcting replication errors. The major conclusions are (i) the mutation rate of a wild-type E. coli strain is ∼1 × 10 −3 per genome per generation; (ii) mutations in the wild-type strain have the expected mutational bias for G:C > A:T mutations, but the bias changes to A:T > G:C mutations in the absence of MMR; (iii) during replication, A:T > G:C transitions preferentially occur with A templating the lagging strand and T templating the leading strand, whereas G:C > A:T transitions preferentially occur with C templating the lagging strand and G templating the leading strand; (iv) there is a strong bias for transition mutations to occur at 5′ApC3′/3′TpG5′ sites (where bases 5′A and 3′T are mutated) and, to a lesser extent, at 5′GpC3′/3′CpG5′ sites (where bases 5′G and 3′C are mutated); (v) although the rate of small (≤4 nt) insertions and deletions is high at repeat sequences, these events occur at only 1/10th the genomic rate of base-pair substitutions. MMR activity is genetically regulated, and bacteria isolated from nature often lack MMR capacity, suggesting that modulation of MMR can be adaptive. Thus, comparing results from the wild-type and MMR-defective strains may lead to a deeper understanding of factors that determine mutation rates and spectra, how these factors may differ among organisms, and how they may be shaped by environmental conditions. evolution | mutation accumulation | neutral mutation | mutational hotspots | indels M utations are the source of variation upon which natural selection acts; thus, a complete understanding of evolutionary processes must include an accurate assessment of mutation rates and of the molecular spectrum of mutational events. In addition, we need to know whether, and how, intrinsic and extrinsic factors influence mutational processes. This understanding must be founded on baseline parameters established by analyzing mutations that accumulate in a neutral fashion, unbiased by selective pressures. Much of our knowledge of spontaneous mutation is based on mutations that occur in nonessential reporter genes during short-term laboratory culture of microorganisms (1). An alternative approach is to compare presumably neutral mutations that have accumulated over evolutionary time periods in diverged species (2). Both methods have substantial uncertainties. The experimental approach may use reporter loci that are not representative of the whole genome and necessarily incorporates assumptions about the expression and neutrality of the mutant phenotypes. The historical approach relies on estimated divergence times and the absence of selective pressure on synonymous sequence changes. High-throughput whole-genome sequencing allows some of these limitations to be...

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Section: Distribution Of Mutations Is Random Among Wild-type Lines Bucontrasting

confidence: 99%

Rate and molecular spectrum of spontaneous mutations in the bacteriumEscherichia colias determined by whole-genome sequencing

Lee¹,

Popodi

Tang³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

648

131

859

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“…The overall rate of BS occurrence was slightly higher (1.8-fold [ Table 2]) in the hightranscription than in the low-transcription strain. The small effect of high levels of transcription on BS mutagenesis detected at LYS2 is consistent with the observed increase in BS in response to transcriptional induction in E. coli (3,24). In the E. coli studies, a specific increase in C3T mutations has been attributed to enhanced deamination of cytosines located on the NTS, presumably because of the transcription-associated single-stranded character of this strand (3).…”

Section: Discussionsupporting

confidence: 78%

“…These data suggest that high levels of transcription do not simply impose additional mutations on top of a basal pattern of mutagenesis, but rather VOL. 24,2004 TRANSCRIPTION-ASSOCIATED MUTATION SPECTRUM IN YEAST 4807 that high transcription alters the basic mechanism(s) of mutagenesis. Concomitant with a transcription-associated decrease in the measured percentage of BS, short Ins-Del mutations comprised a larger percentage of the high-transcription mutations than of the low-transcription mutations (56 and 27%, respectively).…”

Section: Discussionmentioning

confidence: 99%

“…In addition to promoting strand-specific repair, high levels of transcription have been shown to elevate recombination rates in Saccharomyces cerevisiae (33,36,39,41), Schizosaccharomyces pombe (15), and mammalian cells (34). Finally, increased transcription has been shown to stimulate spontaneous mutagenesis in Escherichia coli (3,24,44) and bacteriophage T7 (4) and to enhance deletions within an E. coli plasmid target (40). In S. cerevisiae, Datta and Jinks-Robertson demonstrated previously that an increased transcription level likewise stimulates spontaneous mutation rates, a phenomenon termed transcriptionassociated mutation, or TAM (8).…”

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confidence: 99%

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Identification of a Distinctive Mutation Spectrum Associated with High Levels of Transcription in Yeast

Lippert

Freedman

Barber

et al. 2004

Molecular and Cellular Biology

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High levels of transcription are associated with increased mutation rates in Saccharomyces cerevisiae, a phenomenon termed transcription-associated mutation (TAM). To obtain insight into the mechanism of TAM, we obtained LYS2 forward mutation spectra under low-versus high-transcription conditions in which LYS2 was expressed from either the low-level pLYS2 promoter or the strong pGAL1-10 promoter, respectively. Because of the large size of the LYS2 locus, forward mutations first were mapped to specific LYS2 subregions, and then those mutations that occurred within a defined 736-bp target region were sequenced. In the low-transcription strain base substitutions comprised the majority (64%) of mutations, whereas short insertion-deletion mutations predominated (56%) in the high-transcription strain. Most notably, deletions of 2 nucleotides (nt) comprised 21% of the mutations in the high-transcription strain, and these events occurred predominantly at 5 -(G/C)AAA-3 sites. No ؊2 events were present in the low-transcription spectrum, thus identifying 2-nt deletions as a unique mutational signature for TAM.Transcription influences genomic stability in a complex manner by affecting DNA repair, recombination, mutagenesis, and chromatin structure (reviewed in references 1 and 31). In the subpathway of nucleotide excision repair known as transcription-coupled repair, for example, the encounter of RNA polymerase (Pol) with a transcription-blocking lesion on the transcribed strand specifically triggers repair of the damage, resulting in more efficient repair of lesions on the transcribed strand than on the nontranscribed strand (16). In addition to promoting strand-specific repair, high levels of transcription have been shown to elevate recombination rates in Saccharomyces cerevisiae (33,36,39,41), Schizosaccharomyces pombe (15), and mammalian cells (34). Finally, increased transcription has been shown to stimulate spontaneous mutagenesis in Escherichia coli (3,24,44) and bacteriophage T7 (4) and to enhance deletions within an E. coli plasmid target (40). In S. cerevisiae, Datta and Jinks-Robertson demonstrated previously that an increased transcription level likewise stimulates spontaneous mutation rates, a phenomenon termed transcriptionassociated mutation, or TAM (8). Specifically, reversion of the lys2⌬Bgl frameshift allele and forward mutation at the LYS2 locus increased 35-and 10-fold, respectively, in response to transcriptional induction from the pGAL1-10 promoter. Whereas the genetic requirements and underlying mechanisms of transcription-coupled repair (16, 38) and transcription-associated recombination are becoming clearer (11,12,21), the mechanism of TAM is still poorly understood.The genetic requirements and mutation spectrum of TAM have been characterized in yeast strains using lys2⌬Bgl, a ϩ4 frameshift allele that reverts by acquisition of compensatory Ϫ1 frameshift mutations. In the high-transcription strain, 70% of the reversion mutations required Rev3p, a component of the translesion synthesis DNA Pol , sugg...

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“…1B). Control and AID-treated plasmids were transformed into BW504, a uracil-DNA glycosylase-deficient E. coli strain (17). The transformants were grown on either kanamycin or carbenicillin plates.…”

Section: Aid Canmentioning

confidence: 99%

Activation-induced cytidine deaminase (AID) can target both DNA strands when the DNA is supercoiled

Sui

Storb

2004

Proc. Natl. Acad. Sci. U.S.A.

141

149

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The activation-induced cytidine deaminase (AID) is required for somatic hypermutation (SHM) and class-switch recombination of Ig genes. It has been shown that in vitro, AID protein deaminates C in single-stranded DNA or the coding-strand DNA that is being transcribed but not in double-stranded DNA. However, in vivo, both DNA strands are mutated equally during SHM. We show that AID efficiently deaminates C on both DNA strands of a supercoiled plasmid, acting preferentially on SHM hotspot motifs. However, this DNA is not targeted by AID when it is relaxed after treatment with topoisomerase I, and thus, supercoiling plays a crucial role for AID targeting to this DNA. Most of the mutations are in negatively supercoiled regions, suggesting a mechanism of AID targeting in vivo. During transcription the DNA sequences upstream of the elongating RNA polymerase are negatively supercoiled, and this transient change in DNA topology may allow AID to access both DNA strands.A major advance in the study of somatic hypermutation (SHM) and class-switch recombination (CSR) has been the discovery of the activation-induced cytidine deaminase (AID) (1-3), which appears to be the long-sought SHM mutator factor and inducer of CSR (4). Current in vitro data show that AID is a DNA-specific cytidine deaminase that preferentially removes the amino group of cytidine in single-stranded DNA and in the nontranscribed strand when transcription is active (5-11). These findings are consistent with previous experiments in which SHM is linked to transcription (12, 13). However, in vivo, both DNA strands are equally mutated (14). Furthermore, in ung Ϫ/Ϫ mice, where almost all of the C or G mutations are transitions due to unrepaired AID lesions, equal targeting of both strands is confirmed (15). Single-stranded DNA would also occur in vivo as a potential AID target during DNA replication, which would explain why both DNA strands are targeted during SHM and CSR. However, experiments with a cell line that undergoes SHM in culture support the conclusion that AID can act during the G 1 phase of the cell cycle, and therefore, is not restricted to the S phase (16) (S. Gasior and U.S., unpublished data). A nonexclusive third possibility is that DNA topology (e.g., supercoiling) creates an AID-accessible conformation. In vivo, Ig genes are associated in nucleosomes with histones and other chromatin proteins. These associations, as well as the process of transcription, affect the topology of DNA. To test the possibility that supercoiled DNA as it exists in vivo may be a target for AID, we carried out cytidine deamination assays in vitro with AID purified from insect cells (5). The supercoiled target DNA was an Escherichia coli plasmid that had been manipulated to allow bacterial resistance to carbenicillin only when the initiator AUG of an ampicillin resistance (Amp r ) gene was created by AID deamination of an ACG triplet. Materials and MethodsPlasmid Construction. The kanamycin-resistance (Kan r ) gene was inserted into the SacII site in pBluescript KS(II...

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Transcription-Dependent Increase in Multiple Classes of Base Substitution Mutations in Escherichia coli

Cited by 39 publications

References 21 publications

Rate and molecular spectrum of spontaneous mutations in the bacteriumEscherichia colias determined by whole-genome sequencing

Rate and molecular spectrum of spontaneous mutations in the bacteriumEscherichia colias determined by whole-genome sequencing

Identification of a Distinctive Mutation Spectrum Associated with High Levels of Transcription in Yeast

Activation-induced cytidine deaminase (AID) can target both DNA strands when the DNA is supercoiled

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