scholarly journals Slipped Misalignment Mechanisms of Deletion Formation: In Vivo Susceptibility to Nucleases

1999 ◽  
Vol 181 (2) ◽  
pp. 477-482 ◽  
Author(s):  
Malgorzata Bzymek ◽  
Catherine J. Saveson ◽  
Vladimir V. Feschenko ◽  
Susan T. Lovett
Keyword(s):  
Mismatch Repair ◽  
Dna Polymerase ◽  
Repeated Sequences ◽  
Repair System ◽  
Dna Polymerase Iii ◽  
Exonuclease I ◽  
Polymerase Iii ◽  
Mismatch Repair System ◽  
Genetic Rearrangements

ABSTRACT Misalignment of repeated sequences during DNA replication can lead to deletions or duplications in genomic DNA. In Escherichia coli, such genetic rearrangements can occur at high frequencies, independent of the RecA-homologous recombination protein, and are sometimes associated with sister chromosome exchange (SCE). Two mechanisms for RecA-independent genetic rearrangements have been proposed: simple replication misalignment of the nascent strand and its template and SCE-associated misalignment involving both nascent strands. We examined the influence of the 3′ exonuclease of DNA polymerase III and exonuclease I on deletion via these mechanisms in vivo. Because mutations in these exonucleases stimulate tandem repeat deletion, we conclude that displaced 3′ ends are a common intermediate in both mechanisms of slipped misalignments. Our results also confirm the notion that two distinct mechanisms contribute to slipped misalignments: simple replication misalignment events are sensitive to DNA polymerase III exonuclease, whereas SCE-associated events are sensitive to exonuclease I. If heterologies are present between repeated sequences, the mismatch repair system dependent on MutS and MutH aborts potential deletion events via both mechanisms. Our results suggest that simple slipped misalignment and SCE-associated misalignment intermediates are similarly susceptible to destruction by the mismatch repair system.

scholarly journals Antagonism of Ultraviolet-Light Mutagenesis by the Methyl-Directed Mismatch-Repair System of Escherichia coli

Genetics ◽  
2000 ◽  
Vol 154 (2) ◽  
pp. 503-512 ◽  
Author(s):  
Hongbo Liu ◽  
Stephen R Hewitt ◽  
John B Hays
Keyword(s):  
Escherichia Coli ◽  
Mismatch Repair ◽  
Excision Repair ◽  
Spontaneous Mutation ◽  
Repair System ◽  
Uv Mutagenesis ◽  
Repair Protein ◽  
Mismatch Repair System

Abstract Previous studies have demonstrated that the Escherichia coli MutHLS mismatch-repair system can process UV-irradiated DNA in vivo and that the human MSH2·MSH6 mismatch-repair protein binds more strongly in vitro to photoproduct/base mismatches than to “matched” photoproducts in DNA. We tested the hypothesis that mismatch repair directed against incorrect bases opposite photoproducts might reduce UV mutagenesis, using two alleles at E. coli lacZ codon 461, which revert, respectively, via CCC → CTC and CTT → CTC transitions. F′ lacZ targets were mated from mut+ donors into mutH, mutL, or mutS recipients, once cells were at substantial densities, to minimize spontaneous mutation prior to irradiation. In umu+ mut+ recipients, a range of UV fluences induced lac+ revertant frequencies of 4–25 × 10−8; these frequencies were consistently 2-fold higher in mutH, mutL, or mutS recipients. Since this effect on mutation frequency was unaltered by an Mfd− defect, it appears not to involve transcription-coupled excision repair. In mut+ umuC122::Tn5 bacteria, UV mutagenesis (at 60 J/m2) was very low, but mutH or mutL or mutS mutations increased reversion of both lacZ alleles roughly 25-fold, to 5–10 × 10−8. Thus, at UV doses too low to induce SOS functions, such as Umu2′D, most incorrect bases opposite occasional photoproducts may be removed by mismatch repair, whereas in heavily irradiated (SOS-induced) cells, mismatch repair may only correct some photoproduct/base mismatches, so UV mutagenesis remains substantial.

The Mechanism of Replication of Single-Stranded DNA. VI. Requirements for Ribonucleoside Triphosphates and DNA Polymerase III

1973 ◽  
Vol 51 (12) ◽  
pp. 1588-1597 ◽  
Author(s):  
David T. Denhardt ◽  
Makoto Iwaya ◽  
Grant McFadden ◽  
Gerald Schochetman
Keyword(s):  
Dna Polymerase ◽  
Dna Polymerases ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Dna Polymerase Iii ◽  
Single Stranded Dna ◽  
E Coli ◽  
Polymerase Iii

Evidence is presented that in Escherichia coli made permeable to nucleotides by exposure to toluene, the synthesis of a DNA chain complementary to the infecting single-stranded DNA of bacteriophage [Formula: see text] requires ATP as well as the four deoxyribonucleoside triphosphates. This synthesis results in the formation of the parental double-stranded replicative-form (RF) molecule. The ATP is not required simply to prevent degradation of the ribonucleoside or deoxyribonucleoside triphosphates; it can be partially substituted for by other ribonucleoside triphosphates.No single one of the known E. coli DNA polymerases appears to be uniquely responsible in vivo for the formation of the parental RF. Since [Formula: see text] replicates well in strains lacking all, or almost all, of the in-vitro activities of DNA polymerases I and II, neither of these two enzymes would seem essential; and in a temperature-sensitive E. coli mutant (dnaEts) deficient in DNA polmerase-I activity and possessing a temperature-sensitive DNA polymerase III, the viral single-stranded DNA is efficiently incorporated into an RF molecule at the restrictive temperature. In contrast, both RF replication and progeny single-stranded DNA synthesis are dependent upon DNA polymerase III activity.

scholarly journals Dysfunctional proofreading in the Escherichia coli DNA polymerase III core

2004 ◽  
Vol 384 (2) ◽  
pp. 337-348 ◽  
Author(s):  
Duane A. LEHTINEN ◽  
Fred W. PERRINO
Keyword(s):  
Dna Polymerase ◽  
Gel Filtration ◽  
Exonuclease Activity ◽  
Dna Polymerase Iii ◽  
Wild Type ◽  
Α Subunit ◽  
Polymerase Iii ◽  
Pol Iii

The ε-subunit contains the catalytic site for the 3′→5′ proofreading exonuclease that functions in the DNA pol III (DNA polymerase III) core to edit nucleotides misinserted by the α-subunit DNA pol. A novel mutagenesis strategy was used to identify 23 dnaQ alleles that exhibit a mutator phenotype in vivo. Fourteen of the ε mutants were purified, and these proteins exhibited 3′→5′ exonuclease activities that ranged from 32% to 155% of the activity exhibited by the wild-type ε protein, in contrast with the 2% activity exhibited by purified MutD5 protein. DNA pol III core enzymes constituted with 11 of the 14 ε mutants exhibited an increased error rate during in vitro DNA synthesis using a forward mutation assay. Interactions of the purified ε mutants with the α- and θ-subunits were examined by gel filtration chromatography and exonuclease stimulation assays, and by measuring polymerase/exonuclease ratios to identify the catalytically active ε511 (I170T/V215A) mutant with dysfunctional proofreading in the DNA pol III core. The ε511 mutant associated tightly with the α-subunit, but the exonuclease activity of ε511 was not stimulated in the α–ε511 complex. Addition of the θ-subunit to generate the α–ε511–θ DNA pol III core partially restored stimulation of the ε511 exonuclease, indicating a role for the θ-subunit in co-ordinating the α–ε polymerase–exonuclease interaction. The α–ε511–θ DNA pol III core exhibited a 3.5-fold higher polymerase/exonuclease ratio relative to the wild-type DNA pol III core, further indicating dysfunctional proofreading in the α–ε511–θ complex. Thus the ε511 mutant has wild-type 3′→5′ exonuclease activity and associates physically with the α- and θ-subunits to generate a proofreading-defective DNA pol III enzyme.

scholarly journals In Vivo Protein Interactions within theEscherichia coli DNA Polymerase III Core

1998 ◽  
Vol 180 (6) ◽  
pp. 1563-1566 ◽  
Author(s):  
Piotr Jonczyk ◽  
Adrianna Nowicka ◽  
Iwona J. Fijałkowska ◽  
Roel M. Schaaper ◽  
Zygmunt Cieśla
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Protein Interactions ◽  
Physiological Role ◽  
Dna Polymerase Iii ◽  
Mutator Phenotype ◽  
Α Subunit ◽  
Polymerase Iii ◽  
Pol Iii

ABSTRACT The mechanisms that control the fidelity of DNA replication are being investigated by a number of approaches, including detailed kinetic and structural studies. Important tools in these studies are mutant versions of DNA polymerases that affect the fidelity of DNA replication. It has been suggested that proper interactions within the core of DNA polymerase III (Pol III) of Escherichia colicould be essential for maintaining the optimal fidelity of DNA replication (H. Maki and A. Kornberg, Proc. Natl. Acad. Sci. USA 84:4389–4392, 1987). We have been particularly interested in elucidating the physiological role of the interactions between the DnaE (α subunit [possessing DNA polymerase activity]) and DnaQ (ɛ subunit [possessing 3′→5′ exonucleolytic proofreading activity]) proteins. In an attempt to achieve this goal, we have used theSaccharomyces cerevisiae two-hybrid system to analyze specific in vivo protein interactions. In this report, we demonstrate interactions between the DnaE and DnaQ proteins and between the DnaQ and HolE (θ subunit) proteins. We also tested the interactions of the wild-type DnaE and HolE proteins with three well-known mutant forms of DnaQ (MutD5, DnaQ926, and DnaQ49), each of which leads to a strong mutator phenotype. Our results show that the mutD5 anddnaQ926 mutations do not affect the ɛ subunit-α subunit and ɛ subunit-θ subunit interactions. However, thednaQ49 mutation greatly reduces the strength of interaction of the ɛ subunit with both the α and the θ subunits. Thus, the mutator phenotype of dnaQ49 may be the result of an altered conformation of the ɛ protein, which leads to altered interactions within the Pol III core.

scholarly journals Mismatch Repair in Escherichia coliCells Lacking Single-Strand Exonucleases ExoI, ExoVII, and RecJ

1998 ◽  
Vol 180 (4) ◽  
pp. 989-993 ◽  
Author(s):  
Reuben S. Harris ◽  
Kimberly J. Ross ◽  
Mary-Jane Lombardo ◽  
Susan M. Rosenberg
Keyword(s):  
Mismatch Repair ◽  
Single Strand ◽  
Mutation Rates ◽  
Repair System ◽  
Exonuclease Activity ◽  
Mismatch Repair System ◽  
Genes Encoding ◽  
Null Mutants

ABSTRACT In vitro, the methyl-directed mismatch repair system ofEscherichia coli requires the single-strand exonuclease activity of either ExoI, ExoVII, or RecJ and possibly a fourth, unknown single-strand exonuclease. We have created the first precise null mutations in genes encoding ExoI and ExoVII and find that cells lacking these nucleases and RecJ perform mismatch repair in vivo normally such that triple-null mutants display normal mutation rates. ExoI, ExoVII, and RecJ are either redundant with another function(s) or are unnecessary for mismatch repair in vivo.

scholarly journals INTERACTION OF DNA POLYMERASE III γ AND β SUBUNITS IN VIVO IN SALMONELLA TYPHIMURIUM

Genetics ◽  
1986 ◽  
Vol 113 (3) ◽  
pp. 499-515
Author(s):  
Joyce Engstrom ◽  
Annette Wong ◽  
Russell Maurer
Keyword(s):  
Salmonella Typhimurium ◽  
Dna Polymerase ◽  
Regulatory Gene ◽  
Temperature Sensitive ◽  
Physical Interaction ◽  
Dna Polymerase Iii ◽  
Β Subunit ◽  
Γ Subunit ◽  
Polymerase Iii

ABSTRACT We show that temperature-sensitive mutations in dnaZ, the gene for the γ subunit of DNA polymerase III holoenzyme, can be suppressed by mutations in the dnaN gene, which encodes the β subunit. These results support a direct physical interaction of these two subunits during polymerase assembly or function. The suppressor phenotype is also sensitive to modulation by the dnaA genotype. Since dnaA is organized in an operon with dnaN, and dnaA is a regulatory gene of this operon, we propose that the dnaA effect on suppression can best be explained by modulation of suppressor dnaN levels.

Sign in / Sign up

Export Citation Format

Share Document