Role of Escherichia coli DNA Polymerase IV in In Vivo Replication Fidelity

Abstract

We have investigated whether DNA polymerase IV (Pol IV; the dinB gene product) contributes to the error rate of chromosomal DNA replication in Escherichia coli. We compared mutation frequencies in mismatch repair-defective strains that were either dinB positive or dinB deficient, using a series of mutational markers, including lac targets in both orientations on the chromosome. Virtually no contribution of Pol IV to the chromosomal mutation rate was observed. On the other hand, a significant effect of dinB was observed for reversion of a lac allele when the lac gene resided on an F(pro-lac) episome.Several mechanisms control the fidelity of the DNA replication process. These include correct base selection by the DNA polymerase, removal of base insertion errors by 3Ј-exonucleolytic proofreading, and correction by DNA mismatch repair (29). In Escherichia coli, base selection and proofreading are performed by the DNA polymerase III (Pol III) holoenzyme, the enzyme that replicates the bacterial chromosome. It is generally considered a highly accurate enzyme (29). Mismatch repair is performed by the mutHLS mismatch repair system (17). In combination, these three processes yield an error rate of 10 Ϫ9 to 10 Ϫ11 error per base pair replicated per cell division (6,29).In addition to Pol III, E. coli possesses four other DNA polymerases, Pol I, Pol II, Pol IV, and Pol V, whose precise functions are still being defined. Pol IV and Pol V belong to the recently described Y family of DNA polymerases

Escherichia coli DNA polymerase IV contributes to spontaneous mutagenesis at coding sequences but not microsatellite alleles

“…The overespression of the polymerase causes -1 frameshifts in polypurine runs of G in undamaged DNA [22]. When looking at spontaneous mutagenesis, previous studies have shown a statistically significant decrease in the number of frameshift and base substitution events in the absence of dinB [23,24,32] while overexpression of the dinB gene results in a significant increase in the mutation frequency [21,22]. Pol IV was chosen as a candidate to analyze for strand slippage within microsatellite alleles because a role for the polymerase in spontaneous mutagenesis was acknowledged, as was its role in mutagenesis at homoploymeric G sequences.…”

“…A higher expression of Pol IV is detected from the F factor compared to expression from the chromosomal copy of the dinB gene [24]. Deletion of dinB from the chromosomal location has a negligible effect on mutagenesis [24]; however deletion from the episome produced a significant effect on mutagenesis [24,32]. Pol IV protein levels also are dependent on the growth phase of the culture.…”

“…Many of these studies were performed using a chromosomal target and resulted in no difference in the mutation frequency in the presence of dinB [32,34,35], while a few did observe a decrease in mutagenesis in the absence of dinB [21,23]. One of these studies also examined an episomal mutational target and found that it was more susceptible to mutagenesis than any of the chromosomal targets tested [32]. A study completed previous to this had established that an episomal target was very highly prone to mutagenesis in the presence of dinB [21].…”

Stress responses and genetic variation in bacteria

“…Foster, Submitted for publication). However, loss of Pol IV has little effect on normal, growth-dependent mutation rates, although this also depends on the mutational target [13,15,16] (P.L. Foster, Submitted for publication).…”

“…This had appeared to be true when irrelevant genes, ilvG and rpoB, were monitored for mutation during lactose selection [35,38]. However, the target for Lac + reversion is on F, the conjugal plasmid, and mutation of genes on F appears to occur at a higher rate and by a different mechanism than mutation of genes on the chromosome [16,43,44]. When a second revertible allele, a +1 frameshift in the tetA gene, was on the episome close to the Lac − allele, TetR revertants appeared at about the same rate as did Lac + mutations during lactose selection.…”