DNA Replication Errors Produced by the Replicative Apparatus of Escherichia coli

Fujii, Shingo; Akiyama, Masahiro; Aoki, Kazuhiro; Sugaya, Yutaka; Higuchi, Kiyoshi; Hiraoka, Mina; Miki, Youhei; Saitoh, Naotoshi; Yoshiyama, Kaoru; Ihara, Keiichi; Seki, Mineaki; Ohtsubo, Eiichi; Maki, Hisaji

doi:10.1006/jmbi.1999.2802

Cited by 81 publications

(84 citation statements)

References 44 publications

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“…Products longer than the full-length 30-mer are indicated as ϩ2, ϩ4, ϩ6 etc. 25). 1 In either case, extension of the misaligned primer strand results in expansion of the repeat tract.…”

Section: Figmentioning

confidence: 99%

Dinucleotide Repeat Expansion Catalyzed by Bacteriophage T4 DNA Polymerase in Vitro

Silva¹,

Reha-Krantz

2000

Journal of Biological Chemistry

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DNA replication normally occurs with high fidelity, but certain "slippery" regions of DNA with tracts of mono-, di-, and trinucleotide repeats are frequently mutation hot spots. We have developed an in vitro assay to study the mechanism of dinucleotide repeat expansion. The primer-template resembles a base excision repair substrate with a single nucleotide gap centered opposite a tract of nine CA repeats; nonrepeat sequences flank the dinucleotide repeats. DNA polymerases are expected to repair the gap, but further extension is possible if the DNA polymerase can displace the downstream oligonucleotide. We report here that the wild type bacteriophage T4 DNA polymerase carries out gap and strand displacement replication and also catalyzes a dinucleotide expansion reaction. Repeat expansion was not detected for an exonuclease-deficient T4 DNA polymerase or for Escherichia coli DNA polymerase I. The dinucleotide repeat expansion reaction catalyzed by wild type T4 DNA polymerase required a downstream oligonucleotide to "stall" replication and 3 3 5 exonuclease activity to remove the 3-nonrepeat sequence adjacent to the repeat tract in the template strand. These results suggest that dinucleotide repeat expansion may be stimulated in vivo during DNA repair or during processing of Okazaki fragments.DNA polymerases replicate DNA with high fidelity except for certain DNA sequences, the so-called "slippery" DNAs, which are tracts of simple repeat sequences (reviewed in Ref. 1). The lengths of tracts of mono-, di-, and trinucleotide repeats are unstable, which can easily be detected as repeat-length polymorphisms in microsatellite sequences or as mutation hot spots, such as the classical frameshift hot spots identified by Benzer in the bacteriophage T4 rII genes (2), which are tracts of six A nucleotides (3). Hypermutability in repeat tracts can have serious consequences for human health as observed for an inherited mutation in the human APC gene, which converts the wild type sequence AAATAAAA to the A 8 mononucleotide tract (4). The A 8 sequence was found to create a small hypermutable region for gene inactivating mutations that predispose carriers to colorectal cancer (4). Streisinger et al. (5) suggested that frameshifts are produced in repeat sequences by a transient separation of the primer and template strands and then misalignment during reannealing to generate an intermediate in which one or more repeats is unpaired. This "slippage" intermediate is usually repaired by postreplication mismatch repair as revealed by the dramatic increase in repeat instability when this repair pathway is inactivated (6 -8).In vitro assays have been used to measure DNA polymerasecatalyzed "reiterative replication" of repeat sequences (for examples, see Refs. 9 -14). The number of repeats can be amplified several hundred-fold by a variety of DNA polymerases, but most of the synthetic DNA substrates used are composed exclusively of repeat sequences. One objective of our studies was to develop an improved in vitro assay utilizing a DNA su...

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“…Products longer than the full-length 30-mer are indicated as ϩ2, ϩ4, ϩ6 etc. 25). 1 In either case, extension of the misaligned primer strand results in expansion of the repeat tract.…”

Section: Figmentioning

confidence: 99%

Dinucleotide Repeat Expansion Catalyzed by Bacteriophage T4 DNA Polymerase in Vitro

Silva¹,

Reha-Krantz

2000

Journal of Biological Chemistry

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“…The PCRˆdelity was measured as the mutation frequency in PCR products with the full-length (4.0 kbp) of the plasmid pMOL21, 24) carrying the bla gene for ampicillin resistance and the rpsL gene for the streptomycin-sensitive phenotype, as a template. The plasmid pMOL21 was digested with ScaI at the site on the Amp r gene, and PCR was done using two primers annealed at the ends of the digested plasmid, one of the two primers being biotnylated.…”

Section: Methodsmentioning

confidence: 99%

“…After digestion by the MluI site in the primer region and self-ligation, the reacted products were used to transform the host cells. 24) Colonies formed on the LB plates containing ampicillin were counted as total colonies. The number of colonies formed on the LB plates containing both ampicillin and streptomycin were counted as mutated colonies, because the mutations in the rpsL gene on PCR would allow these cells to grow on the plates.…”

Section: Methodsmentioning

confidence: 99%

Gene Cloning and Polymerase Chain Reaction with Proliferating Cell Nuclear Antigen fromThermococcus kodakaraensisKOD1

Kitabayashi

Nishiya

Esaka

et al. 2002

Bioscience, Biotechnology, and Biochemistry

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“…We have previously proposed a hypothesis that the majority of spontaneous mutations are not derived from replication errors but caused by other premutagenic DNA lesions that are never or inefficiently corrected by the mismatch repair system (Fujii et al, 1999;Yoshiyama et al, 2001;Yoshiyama and Maki 2003). This is based on our findings that patterns of spontaneous mutations occurring in a wild-type Escherichia coli strain were dissimilar to those in a mismatch-repair (MMR) deficient strain, in which replication errors are not corrected and turn into the mutations.…”

Section: Introductionmentioning

confidence: 99%

Roles of RecA protein in spontaneous mutagenesis in Escherichia coli

et al. 2007

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To verify the extent of contribution of spontaneous DNA lesions to spontaneous mutagenesis, we have developed a new genetic system to examine simultaneously both forward mutations and recombination events occurring within about 600 base pairs of a transgenic rpsL target sequence located on Escherichia coli chromosome. In a wild-type strain, the recombination events were occurring at a frequency comparable to that of point mutations within the rpsL sequence. When the cells were UV-irradiated, the recombination events were induced much more sharply than point mutations. In a recA null mutant, no recombination event was observed. These data suggest that the blockage of DNA replication, probably caused by spontaneous DNA lesions, occurs often in normally growing E. coli cells and is mainly processed by cellular functions requiring the RecA protein. However, the recA mutant strain showed elevated frequencies of single-base frameshifts and large deletions, implying a novel mutator action of this strain. A similar mutator action of the recA mutant was also observed with a plasmid-based rpsL mutation assay. Therefore, if the recombinogenic problems in DNA replication are not properly processed by the RecA function, these would be a potential source for mutagenesis leading to single-base frameshift and large deletion in E. coli. Furthermore, the single-base frameshifts induced in the recA-deficient cells appeared to be efficiently suppressed by the mutS-dependent mismatch repair system. Thus, it seems likely that the single-base frameshifts are derived from slippage errors that are not directly caused by DNA lesions but made indirectly during some kind of error-prone DNA synthesis in the recA mutant cells.

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DNA Replication Errors Produced by the Replicative Apparatus of Escherichia coli

Cited by 81 publications

References 44 publications

Dinucleotide Repeat Expansion Catalyzed by Bacteriophage T4 DNA Polymerase in Vitro

Dinucleotide Repeat Expansion Catalyzed by Bacteriophage T4 DNA Polymerase in Vitro

Gene Cloning and Polymerase Chain Reaction with Proliferating Cell Nuclear Antigen fromThermococcus kodakaraensisKOD1

Roles of RecA protein in spontaneous mutagenesis in Escherichia coli

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