dnaC mutations suppress defects in DNA replication- and recombination-associated functions in priB and priC double mutants in Escherichia coli K-12

PriA, PriB and PriC were originally discovered as proteins essential for th e Phi X174 in vitro DNA replication system. Recent studies have shown that PriA mutants are poorly viable, have high basal levels of SOS expression (S OSH), are recombination deficient (Rec(-)), sensitive to UV irradiation (UV S) 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 a nd homologous recombination, Based on the Phi X174 system, mutations in pri B and priC should cause phenotypes like those seen in priA2::kan mutants. T o test this, mutations in priB and priC were constructed. We found that, co ntrary to the Phi X174 model, del(priB)302 and priC303::kan mutants have al most 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 redundan t and important role in Escherichia coil, The priA2::kan suppressor, dnaC80 9, partially suppressed the poor viability/slow growth phenotype of the pri BC double mutant, The resulting triple mutant (priBC dnaC809) had small col ony 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 dna C809 (E176G)]. Experiments suggest that dnaC820 makes dnaC809 suppression o f priA and or priBC mutants priB and or priC independent, A model is propos ed for the roles of these proteins in terms of restarting collapsed replica tion forks from recombinational intermediates.