PMID:10540288

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
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Main Points of the Paper

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Materials and Methods Used

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Phenotype Annotations

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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
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
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
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
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