PMID:10540288

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Citation

Sandler, SJ, Marians, KJ, Zavitz, KH, Coutu, J, Parent, MA and Clark, AJ (1999) dnaC mutations suppress defects in DNA replication- and recombination-associated functions in priB and priC double mutants in Escherichia coli K-12.Mol. Microbiol. 34:91-101

Abstract

PriA, PriB and PriC were originally discovered as proteins essential for the PhiX174 in vitro DNA replication system. Recent studies have shown that PriA mutants are poorly viable, have high basal levels of SOS expression (SOSH), are recombination deficient (Rec-), sensitive to UV irradiation (UVS) and sensitive to rich media. These data suggest that priA's role may be more complex than previously thought and may involve both DNA replication and homologous recombination. Based on the PhiX174 system, mutations in priB and priC should cause phenotypes like those seen in priA2:kan mutants. To test this, mutations in priB and priC were constructed. We found that, contrary to the PhiX174 model, del(priB)302 and priC303:kan mutants have almost wild-type phenotypes. Most unexpectedly, we then found that the priBC double mutant had very poor viability and/or a slow growth rate (even less than a priA2:kan mutant). This suggests that priB and priC have a redundant and important role in Escherichia coli. The priA2:kan suppressor, dnaC809, partially suppressed the poor viability/slow growth phenotype of the priBC double mutant. The resulting triple mutant (priBC dnaC809 ) had small colony size, recombination deficiency and levels of SOS expression similar to a priA2:kan mutant. The priBC dnaC809 mutant, however, was moderately UVR and had good viability, unlike a priA2:kan mutant. Additional mutations in the triple mutant were selected to suppress the slow growth phenotype. One suppressor restored all phenotypes tested to nearly wild-type levels. This mutation was identified as dnaC820 (K178N) [mapping just downstream of dnaC809 (E176G)]. Experiments suggest that dnaC820 makes dnaC809 suppression of priA and or priBC mutants priB and or priC independent. A model is proposed for the roles of these proteins in terms of restarting collapsed replication forks from recombinational intermediates.

Links

PubMed

Keywords

Bacterial Proteins; Bacteriophage mu; Cell Division; DNA Replication; DNA, Bacterial; DNA-Binding Proteins; Escherichia coli; Escherichia coli Proteins; Mutation; Phenotype; Recombination, Genetic; Replication Protein A; SOS Response (Genetics); Substrate Specificity; Suppression, Genetic

Main Points of the Paper

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Materials and Methods Used

Please list the materials and methods used in this paper (strains, plasmids, antibodies, etc).

Phenotype Annotations

See Help:AnnotationTable for details on how to edit this table.

Phenotype of Taxon Information Genotype Information (if known) Condition Information OMP ID OMP Term Name ECO ID ECO Term Name Notes Status

a mutation or genetic difference within a strain

  • Taxon: Escherichia coli
  • Strain: K-12
  • Substrain: DM4000
  • NCBI Taxon ID: 83333
  • Genotype of Reference Strain: priB+
  • Genotype of Experimental Strain : priB302(del)
  • Reference Condition:
OMP:0005056

decreased frequency of site-specific recombination

Reference Table 1; P1 Transduction evidence

needs eco and ref condition

a mutation or genetic difference within a strain

  • Taxon: Escherichia coli
  • Strain: K-12
  • Substrain: DM4000
  • NCBI Taxon ID: 83333
  • Genotype of Reference Strain: priB+
  • Genotype of Experimental Strain : priB302(del)
  • Reference Condition: 20 J UV compared to 0 J
OMP:0005084

UV radiation resistance phenotype

ECO:0000095

cell growth assay evidence

Reference Table 1; phenotype is unchanged

a mutation or genetic difference within a strain

  • Taxon: Escherichia coli
  • Strain: K-12
  • Substrain: DM4000
  • NCBI Taxon ID: 83333
  • Genotype of Reference Strain: priB+
  • Genotype of Experimental Strain : priB302(del)
  • Reference Condition: UV-irradiation at .5 J/m^2/sec for 10 seconds
OMP:0005023

presence of an SOS response

ECO:0000297

LacZ transcript localization evidence

Reference Table 1; phenotype is unchanged

a mutation or genetic difference within a strain

  • Taxon: Escherichia coli
  • Strain: K-12
  • Substrain: DM4000
  • NCBI Taxon ID: 83333
  • Genotype of Reference Strain: priC+
  • Genotype of Experimental Strain : priC303::kan
  • Reference Condition:
OMP:0005058

presence of site-specific recombination

Reference Table 1; P1 Transduction evidence

needs eco and ref condition

a mutation or genetic difference within a strain

  • Taxon: Escherichia coli
  • Strain: K-12
  • Substrain: DM4000
  • NCBI Taxon ID: 83333
  • Genotype of Reference Strain: priC+
  • Genotype of Experimental Strain : priC303::kan
  • Reference Condition: 20 J UV compared to 0 J
OMP:0005084

UV radiation resistance phenotype

ECO:0000095

cell growth assay evidence

Reference Table 1; phenotype is unchanged

a mutation or genetic difference within a strain

  • Taxon: Escherichia coli
  • Strain: K-12
  • Substrain: DM4000
  • NCBI Taxon ID: 83333
  • Genotype of Reference Strain: priC+
  • Genotype of Experimental Strain : priC303::kan
  • Reference Condition: UV-irradiation at .5 J/m^2/sec for 10 seconds
OMP:0005023

presence of an SOS response

ECO:0000297

LacZ transcript localization evidence

Reference Table 1; phenotype is unchanged

a mutation or genetic difference within a strain

  • Taxon: Escherichia coli
  • Strain: K-12
  • Substrain: DM4000
  • NCBI Taxon ID: 83333
  • Genotype of Reference Strain: priA+
  • Genotype of Experimental Strain : priA2::kan
  • Reference Condition:
OMP:0005056

decreased frequency of site-specific recombination

Reference Table 1; P1 Transduction evidence

needs eco and ref condition

a mutation or genetic difference within a strain

  • Taxon: Escherichia coli
  • Strain: K-12
  • Substrain: DM4000
  • NCBI Taxon ID: 83333
  • Genotype of Reference Strain: priA+
  • Genotype of Experimental Strain : priA2::kan
  • Reference Condition: 20 J UV compared to 0 J
OMP:0005098

decreased resistance to UV radiation

ECO:0000095

cell growth assay evidence

Reference Table 1

a mutation or genetic difference within a strain

  • Taxon: Escherichia coli
  • Strain: K-12
  • Substrain: DM4000
  • NCBI Taxon ID: 83333
  • Genotype of Reference Strain: priA+
  • Genotype of Experimental Strain : priA2::kan
  • Reference Condition: UV-irradiation at .5 J/m^2/sec for 10 seconds
OMP:0005025

increased induced expression of the SOS response

ECO:0000297

LacZ transcript localization evidence

Reference Table 1

a mutation or genetic difference within a strain

  • Taxon: Escherichia coli
  • Strain: K-12
  • Substrain: DM4000
  • NCBI Taxon ID: 83333
  • Genotype of Reference Strain: priB+ priC+
  • Genotype of Experimental Strain : priB302(del) priC303::kan
  • Reference Condition: minimal media containing 50 micrograms ml^-1 kanamycin, 15 micrograms ml^-1 X-gal, incubation 3-4 days
OMP:0005020

increased basal expression of the SOS response

ECO:0000297

lacZ transcript localization evidence

Reference section 2 of results section

a mutation or genetic difference within a strain

  • Taxon: Escherichia coli
  • Strain: K-12
  • Substrain: DM4000
  • NCBI Taxon ID: 83333
  • Genotype of Reference Strain: priB+ priC+ dnaC+
  • Genotype of Experimental Strain : priB302(del) priC303::kan dnaC809
  • Reference Condition:
OMP:0005056

decreased frequency of site-specific recombination

Reference Table 1; P1 Transduction evidence

needs eco and ref condition DELETE??

a mutation or genetic difference within a strain

  • Taxon: Escherichia coli
  • Strain: K-12
  • Substrain: DM4000
  • NCBI Taxon ID: 83333
  • Genotype of Reference Strain: priB+ priC+ dnaC+
  • Genotype of Experimental Strain : priB302(del) priC303::kan dnaC809
  • Reference Condition: 20 J UV compared to 0 J
OMP:0005098

decreased resistance to UV radiation

ECO:0000095

cell growth assay evidence

Reference Table 1; phenotype is unchanged

DELETE??

a mutation or genetic difference within a strain

  • Taxon: Escherichia coli
  • Strain: K-12
  • Substrain: DM4000
  • NCBI Taxon ID: 83333
  • Genotype of Reference Strain: priB+ priC+ dnaC+
  • Genotype of Experimental Strain : priB302(del) priC303::kan dnaC809
  • Reference Condition: UV-irradiation at .5 J/m^2/sec for 10 seconds
OMP:0005020

increased basal expression of the SOS response

ECO:0000297

LacZ transcript localization evidence

Reference Table 1; phenotype is unchanged

DELETE??

a mutation or genetic difference within a strain

  • Taxon: Eschericia coli
  • Strain: K-12
  • Substrain: DM4000
  • NCBI Taxon ID: 83333
  • Genotype of Reference Strain: priB302(del) priC303::kan dnaC+
  • Genotype of Experimental Strain : priB302(del) priC303::kan dnaC809,820
  • Reference Condition: 20 J UV compared to 0 J
OMP:0005097

increased resistance to UV radiation

ECO:0000095

Cell growth assay evidence

Reference Table 1; Restores UV resistance completely compared to wild type

a mutation or genetic difference within a strain

  • Taxon: Eschericia coli
  • Strain: K-12
  • Substrain: DM4000
  • NCBI Taxon ID: 83333
  • Genotype of Reference Strain: priB302(del) priC303::kan dnaC+
  • Genotype of Experimental Strain : priB302(del) priC303::kan dnaC809,820
  • Reference Condition: UV-irradiation at .5 J/m^2/sec for 10 seconds
OMP:0005025

decreased induced expression of the SOS response

ECO:0000297

lacZ transcript localization evidence

Reference Table 1; Restores SOS response expression completely compared to wild type

a mutation or genetic difference within a strain

  • Taxon: Eschericia coli
  • Strain: K-12
  • Substrain: DM4000
  • NCBI Taxon ID: 83333
  • Genotype of Reference Strain: priB302(del) priC303::kan dnaC+
  • Genotype of Experimental Strain : priB302(del) priC303::kan dnaC809,820
  • Reference Condition:
OMP:0005055

increased frequency of site-specific recombination

Reference Table 1; Restores recombination frequency to about half of the wild types recombination frequency

needs eco and ref condition

a mutation or genetic difference within a strain

  • Taxon: Escherichia coli
  • Strain: K-12
  • Substrain: DM4000
  • NCBI Taxon ID: 83333
  • Genotype of Reference Strain: priA+ priB+ priC+ dnaC+
  • Genotype of Experimental Strain : priA::kan priB302(del) priC303::kan dnaC809
  • Reference Condition: 20 J UV compared to 0 J
OMP:0005098

decreased resistance to UV radiation

ECO:0000095

cell growth assay evidence

Reference Table 1; Evidence that PriA and DnaC809 do not compete for the same substrate

a mutation or genetic difference within a strain

  • Taxon: Escherichia coli
  • Strain: K-12
  • Substrain: DM4000
  • NCBI Taxon ID: 83333
  • Genotype of Reference Strain: priA+ priB+ priC+ dnaC+
  • Genotype of Experimental Strain : priA::kan priB302(del) priC303::kan dnaC809
  • Reference Condition: UV-irradiation at .5 J/m^2/sec for 10 seconds
OMP:0005020

increased basal expression of the SOS response

ECO:0000297

lacZ transcript localization evidence

Reference Table 1; Evidence that PriA and DnaC809 do not compete for the same substrate

a mutation or genetic difference within a strain

  • Taxon: Escherichia coli
  • Strain: K-12
  • Substrain: DM4000
  • NCBI Taxon ID: 83333
  • Genotype of Reference Strain: priA+ priB+ priC+ dnaC+
  • Genotype of Experimental Strain : priA::kan priB302(del) priC303::kan dnaC809
  • Reference Condition:
OMP:0005056

decreased frequency of site-specific recombination

Reference Table 1; Evidence that PriA and DnaC809 do not compete for the same substrate

Needs eco and ref condition

a mutation or genetic difference within a strain

  • Taxon: Escherichia coli
  • Strain: K-12
  • Substrain: DM4000
  • NCBI Taxon ID: 83333
  • Genotype of Reference Strain: priA2::kan dnaC+
  • Genotype of Experimental Strain : priA2::kan dnaC809, 820
  • Reference Condition:
OMP:0005055

increased frequency of site-specific recombination

Reference Table 1; Restores recombination fully compared to wild type; P1 Transduction evidence

needs eco and ref condition

a mutation or genetic difference within a strain

  • Taxon: Escherichia coli
  • Strain: K-12
  • Substrain: DM4000
  • NCBI Taxon ID: 83333
  • Genotype of Reference Strain: priA2::kan dnaC+
  • Genotype of Experimental Strain : priA2::kan dnaC809, 820
  • Reference Condition: 20 J UV compared to 0 J
OMP:0005097

increased resistance to UV radiation

ECO:0000095

cell growth assay evidence

Reference Table 1; Restored almost all of UV radiation resistance compared to wild type

a mutation or genetic difference within a strain

  • Taxon: Escherichia coli
  • Strain: K-12
  • Substrain: DM4000
  • NCBI Taxon ID: 83333
  • Genotype of Reference Strain: priA2::kan dnaC+
  • Genotype of Experimental Strain : priA2::kan dnaC809, 820
  • Reference Condition: UV-irradiation at .5 J/m^2/sec for 10 seconds
OMP:0005025

decreased induced expression of the SOS response

ECO:0000297

LacZ transcript localization evidence

Reference Table 1; Expression still nearly twice that of the wild type

a mutation or genetic difference within a strain

  • Taxon: Escherichia coli
  • Strain: K-12
  • Substrain: DM4000
  • NCBI Taxon ID: 83333
  • Genotype of Reference Strain: priB302(del) priC303::kan dnaC+
  • Genotype of Experimental Strain : priB302(del) priC303::kan dnaC809, 820
  • Reference Condition:

a mutation or genetic difference within a strain

  • Taxon: Escherichia coli
  • Strain: K-12
  • Substrain: DM4000
  • NCBI Taxon ID: 83333
  • Genotype of Reference Strain: priB302(del) priC303::kan dnaC+
  • Genotype of Experimental Strain : priB302(del) priC303::kan dnaC809, 820
  • Reference Condition:

a mutation or genetic difference within a strain

  • Taxon: Escherichia coli
  • Strain: K-12
  • Substrain: DM4000
  • NCBI Taxon ID: 83333
  • Genotype of Reference Strain: priB302(del) priC303::kan dnaC+
  • Genotype of Experimental Strain : priB302(del) priC303::kan dnaC809, 820
  • Reference Condition:

a mutation or genetic difference within a strain

  • Taxon: Escherichia coli
  • Strain: K-12
  • Substrain: DM4000
  • NCBI Taxon ID: 83333
  • Genotype of Reference Strain:
  • Reference Condition:



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References

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