DNA polymerase I and the bypassing of RecA dependence of constitutive stable DNA replication in Escherichia coli rnhA mutants

Abstract

In Escherichia coli rnhA mutants, several normally repressed origins (oriK sites) of DNA replication are activated. The type of DNA replication initiated from these origins, termed constitutive stable DNA replication, does not require DnaA protein or the oriC site, which are essential for normal DNA replication. It requires active RecA protein. We previously found that the lexA71(Def)::Tn5 mutation can suppress this RecA requirement and postulated that the derepression of a LexA regulon gene(s) leads to the activation of a bypass pathway, Rip (for RecA-independent process). In this study, we isolated a miniTn10spc insertion mutant that abolishes the ability of the lexA(Def) mutation to suppress the RecA requirement of constitutive stable DNA replication. Cloning and DNA sequencing analysis of the mutant revealed that the insertion occurs at the 3' end of the coding region of the polA gene, which encodes DNA polymerase I. The mutant allele, designated polA25::miniTn10spc, is expected to abolish the polymerization activity but not the 5'-->3' or 3'-->5' exonuclease activity. Thus, the Rip bypass pathway requires active DNA polymerase I. Since the lethal combination of recA(Def) and polA25::miniTn10spc could be suppressed by derepression of the LexA regulon only when DNA replication is driven by the oriC system, it was suggested that the bypass pathway has a specific requirement for DNA polymerase I at the initiation step in the absence of RecA. An accompanying paper (Y. Cao and T. Kogoma, J. Bacteriol. 175:7254-7259, 1993) describes experiments to determine which activities of DNA polymerase I are required at the initiation step and discusses possible roles for DNA polymerase in the Rip bypass pathway.

DNA polymerase I in constitutive stable DNA replication in Escherichia coli

“…The data for AQ634, AQ10810, AQ10850, and AQ10814 are averages (Ϯ standard errors of means) of four, two, three, and two independent determinations, respectively. (6). In this study, we found that the polA25::miniTn10spc mutation inactivates cSDR in recA ϩ strains in a temperature-dependent manner ( Fig.…”

“…The requirement for RecA in cSDR can be bypassed by derepression of the LexA regulon by introduction of a lexA(Def) mutation, leading to activation of the Rip (RecA-independent process) bypass pathway (6,48). Thus, dnaA::Tn10 rnhA102 recA(Ts) lexA(Def) quadruple mutants are viable at 42ЊC despite the nonfunctional oriC system due to dnaA::Tn10 and despite the inactivation of the oriK system by recA(Ts) at the restrictive temperature.…”

“…polA1 is an amber mutation, and this allele encodes a truncated protein which has little pol activity but possesses a nearly normal level of the 5Ј33Ј exo activity (40). The polA25::miniTn10spc allele has a miniTn10 insertion at the end of the coding region and encodes a truncated protein missing the C-terminal 57 amino acid residues (6). This portion of the enzyme includes the last ␣ helix and three ␤ sheets which contain at least two residues crucial for the pol activity (42).…”

Activation of the Rcs Signal Transduction System Is Responsible for the Thermosensitive Growth Defect of anEscherichia coliMutant Lacking Phosphatidylglycerol and Cardiolipin

“…DNA procedures were based on standard methods (38,46). Mutagenesis with mini-Tn10 cam (30) was performed essentially as described by Cao et al (4). Briefly, strain S330(pNK2884) grown to early exponential phase was incubated for 30 min with 0.1 mM isopropyl-␤-Dthiogalactopyranoside (IPTG) to induce the expression of transposase.…”

D-loops and R-loops: alternative mechanisms for the initiation of chromosome replication in Escherichia coli

“…Thus, derepression of one or more LexA regulon genes activates a bypass pathway which functions in the absence of RecA (9). Interestingly, the bypass requires the nick translation activity of DNA Pol l (8).…”