Roles of chromosomal and episomal dinB genes encoding DNA pol IV in targeted and untargeted mutagenesis in Escherichia coli

Abstract

DNA polymerase IV (pol IV) in Escherichia coli is a member of a novel family of DNA polymerases (the DinB/UmuC/Rad30/Rev1 super-family or the DNA polymerase Y family). Although expression of the dinB gene encoding DNA pol IV is known to result in an enhancement of untargeted mutagenesis, it remains uncertain whether DNA pol IV is involved in a variety of lesion-induced mutagenesis (targeted mutagenesis), and the relationship between expression levels of dinB and the mutagenesis that DNA pol IV promotes has not been investigated thoroughly. Here, we report that DNA pol IV is involved in -1 frameshift mutagenesis induced by 4-nitroquinoline N-oxide (4-NQO) and that the expression level of the chromosomal pol IV gene is 6-12 times higher than those for other SOS-inducible DNA polymerases in E. coli, i.e., DNA pol II (PolB) or DNA pol V (UmuDC), respectively. Interestingly, the dinB gene is present not only on the chromosome but also on the F' plasmid in the E. coli CC108 strain. In this strain, 750 molecules of DNA pol IV are expressed from the F' dinB gene in the uninduced state and 250 molecules are expressed from the chromosomal gene. These cellular expression levels strongly affect -1 frameshifts induced by 4-NQO in runs of six guanine bases: mutagenicity was highest in the strain CC108, followed by strains YG2242 (chromosome deltadinB/F' dinB+), YG2247 (chromosome dinB+/F' deltadinB) and FC1243 (chromosome deltadinB/F' deltadinB). The incidence of untargeted -1 frameshifts was reduced by two-thirds on deletion of dinB from the F' episome. The chromosomal dinB gene appeared to have little or no effect on the untargeted mutagenesis. These results suggest that DNA pol IV efficiently mediates targeted mutagenesis by 4-NQO, and that the cellular levels of expression substantially affect targeted and untargeted mutagenesis.

Interactions and Localization of Escherichia coli Error-Prone DNA Polymerase IV after DNA Damage

“…Pol IV appears to be specialized for bypassing adducts to the N 2 position of guanines, such as benzo(␣)pyrene, nitrofurazone, and 4-nitroquinoline-1-oxide (NQO) (3,7,8). Pol IV is relatively abundant (250 molecules per cell) even in non-SOS-induced cells (9). These high levels suggest that Pol IV may gain access to replicating DNA, and indeed, Pol IV can displace the replicase DNA polymerase III from the sliding clamp in vitro (10,11).…”

“…These phenotypes suggest an important role for Pol IV in rescuing stalled replication forks during replication. After SOS induction, the levels of Pol IV increase 10-fold to about 2,500 molecules per cell (9). In stationary-phase cells, Pol IV is induced 3-fold under the control of the general stress response sigma factor RpoS (13).…”

The Roles of UmuD in Regulating Mutagenesis

“…LexA cleavage inactivates it as a repressor and exposes a proteolysis signal sequence, leading to degradation of LexA [24] and to increased expression of at least 57 SOS-regulated genes, including UmuD [20]. The cellular levels of UmuD, UmuC, and DinB all increase approximately 10-fold upon SOS induction, with UmuD increasing from ~180 to ~2400 molecules, UmuC increasing from ~15 to ~200 molecules, and DinB increasing from ~250 to ~2500 molecules per cell [25, 26]. The products of SOS-regulated genes are involved in DNA repair, DNA damage tolerance, and regulation of cell division.…”

“…DinB (Pol IV) is the other Y-family lesion bypass polymerase in E. coli and is the only Y-family polymerase that is conserved throughout all domains of life [5, 15]. The expression level of chromosomal DinB under DNA damaging conditions is 6–12 times higher than that of UmuC or PolB (DNA pol II) with about 2500 molecules of DinB in an SOS-induced cell [25]. DinB is also found on the recombinant F′ plasmid that was constructed to determine mutation spectra of specific revertible l a c − alleles [25, 93].…”

“…The expression level of chromosomal DinB under DNA damaging conditions is 6–12 times higher than that of UmuC or PolB (DNA pol II) with about 2500 molecules of DinB in an SOS-induced cell [25]. DinB is also found on the recombinant F′ plasmid that was constructed to determine mutation spectra of specific revertible l a c − alleles [25, 93]. The expression level of DinB in an uninduced state from the F′ plasmid in E. coli strain CC108 is approximately 750 molecules, as compared to 250 molecules expressed from the chromosome in the absence of SOS induction [25].…”