Polymerases Leave Fingerprints: Analysis of the Mutational Spectrum in Escherichia coli rpoB To Assess the Role of Polymerase IV in Spontaneous Mutation

Abstract

We compared the distribution of mutations in rpoB that lead to rifampin resistance in strains with differing levels of polymerase IV (Pol IV), including strains with deletions of the Pol IV-encoding dinB gene, strains with a chromosomal copy of dinB, strains with the F128 plasmid, and strains with plasmid amplification of either the dinB operon (dinB-yafNOP) or the dinB gene alone. This analysis identifies several hot spots specific to Pol IV which are virtually absent from the normal spontaneous spectrum, indicating that Pol IV does not contribute significantly to mutations occurring during exponential growth in liquid culture.Damage-inducible polymerases (20, 22; for reviews, see references 9 and 16), such as the SOS-induced polymerase IV (Pol IV) and Pol V in Escherichia coli, not only bypass certain noncoding lesions but also increase replication errors across from normal bases (19,20,23). Their discovery has led to the suggestion that a significant fraction of spontaneous mutations in growing cells under normal conditions might be due to errors caused by basal levels of error-prone polymerases (18). The dinB-encoded Pol IV is the leading candidate, since the overexpression of dinB on high-copy plasmids leads to increases in base substitutions and frameshifts, particularly Ϫ1 frameshifts (11,12,23). Moreover, several studies have shown an approximately twofold decrease in spontaneous mutations in strains with an inactivating allele of dinB that also reduces the expression of three genes downstream of dinB-yafNOP (14, 18), although this effect is not present if only dinB is inactivated (14). The expression of dinB and yafNOP is increased after SOS induction by DNA-damaging agents (4), and these four genes have been shown to be part of an operon (14).We decided to examine the spectra of base substitution mutations in strains with differing levels of dinB expression, since a comparison of detailed genetic fingerprints of these strains might reveal patterns specific to processes involving and not involving Pol IV. We recently characterized a system using mutations in the rpoB gene that yield the rifampin resistance (Rif r ) phenotype at 37°C in order to analyze the base substitution profiles of mutagens and mutators (10). We have now characterized 77 mutations in rpoB. Each of the six base substitutions is monitored with a set of 9 to 17 sites. In the study reported here, we looked at cells that carry a single copy of the dinB operon on the chromosome and compared the mutational spectrum of these cells with those of strains with deletions of the dinB gene, strains that carry a second copy of the dinB operon on an FЈ plasmid, and strains that carry a multicopy plasmid with an insert containing the dinB operon in one case and just the dinB gene in another case. We showed that some mutational hot spots are specific for the overexpression of the dinB operon and that others are found in the spectrum of wild-type strains but not after the amplification of the dinB operon. A comparison of the different spectra leads us...

Rate and molecular spectrum of spontaneous mutations in the bacteriumEscherichia colias determined by whole-genome sequencing

“…As mentioned above, A:T transversions occurred preferentially at GATC sites where the A is methylated at the 6 position by the Dam methylase. GATC sites also were hotspots in the mutational spectrum of the rpoB gene (69). Depurinated bases lead to transversion mutations because for many DNA polymerases the preferred order of base insertion opposite a missing base is A followed by G, although this order is far from strict (33,70).…”

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

“…Pol IV protein levels also are dependent on the growth phase of the culture. Levels of Pol IV protein are transcriptionally induced in late stationary phase [33], and Pol IV does not contribute to spontaneous mutations during exponential growth when expressed at endogenous levels [34].…”

“…There are many reports in the literature in which both chromosomally and episomally located targets were used to look at a role for Pol IV in mutagenesis. 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].…”

An editing-defective aminoacyl-tRNA synthetase is mutagenic in aging bacteria via the SOS response

“…In particular, Rif R mutations typically fall in one of three clusters of rpoB. These regions have been extensively sequenced and characterized in earlier work (10)(11)(12). To examine the mutations in detail, 48 clonal DNA isolates were sequenced for each genotype (wild-type and ileS Ala ) after each time period (1 and 7 days).…”

“…Certain mechanisms of mutation are known to result in ''signatures,'' where only specific types of genetic changes occur (11,12). The GC 3 AT mutation is a signature for DNA-repair- Fig.…”