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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...

SummaryWe searched for genes that create mutator phenotypes when put on to a multicopy plasmid in Escherichia coli . In many cases, this will result in overexpression of the gene in question. We constructed a random shotgun library with E. coli genomic fragments between 3 and 5 kbp in length on a multicopy plasmid vector that was transformed into E. coli to screen for frameshift mutators. We identified a total of 115 independent genomic fragments that covered 17 regions on the E. coli chromosome. Further studies identified 12 genes not previously known as causing mutator phenotypes when overproduced. A striking finding is that overproduction of the multidrug resistance transcription regulator, EmrR, results in a large increase in frameshift and base substitution mutagenesis. This suggests a link between multidrug resistance and mutagenesis. Other identified genes include those encoding DNA helicases (UvrD, RecG, RecQ), truncated forms of the DNA mismatch repair protein (MutS) and a primosomal component (DnaT), a negative modulator of initiation of replication/GATCbinding protein (SeqA), a stationary phase regulator AppY, a transcriptional regulator PaaX and three putative open reading frames, ycgW , yfjY and yjiD , encoding hypothetical proteins. In addition, we found three genes encoding proteins that were previously known to cause mutator effects under overexpression conditions: error-prone polymerase IV (DinB), DNA methylase (Dam) and sigma S factor (RpoS). This genomic strategy offers an approach to identify novel mutator effects resulting from the multicopy cloning (MCC) of specific genes and therefore complementing the conventional gene inactivation approach to finding mutators.

We show that the MutY protein competes with the MutS-dependent mismatch repair system to process at least some A ⅐ C mispairs in vivo, converting them to G ⅐ C pairs. In the presence of an increased dCTP pool resulting from the loss of nucleotide diphosphate kinase, the frequency of A ⅐ T3G ⅐ C transitions at a hot spot in the rpoB gene is 30-fold lower in a MutY-deficient derivative than in the wild type.

We have applied a genetic system for analyzing mutations in Escherichia coli to Deinococcus radiodurans, an extremeophile with an astonishingly high resistance to UV-and ionizing-radiation-induced mutagenesis. Taking advantage of the conservation of the ␤-subunit of RNA polymerase among most prokaryotes, we derived again in D. radiodurans the rpoB/Rif r system that we developed in E. coli to monitor base substitutions, defining 33 base change substitutions at 22 different base pairs. We sequenced Ͼ250 mutations leading to Rif r in D. radiodurans derived spontaneously in wild-type and uvrD (mismatch-repair-deficient) backgrounds and after treatment with N-methyl-N Ј-nitro-N-nitrosoguanidine (NTG) and 5-azacytidine (5AZ). The specificities of NTG and 5AZ in D. radiodurans are the same as those found for E. coli and other organisms. There are prominent base substitution hotspots in rpoB in both D. radiodurans and E. coli. In several cases these are at different points in each organism, even though the DNA sequences surrounding the hotspots and their corresponding sites are very similar in both D. radiodurans and E. coli. In one case the hotspots occur at the same site in both organisms.A S we continue to explore the vast diversity of microtwo small regions of rpoB, they can be analyzed by using only two primer pairs for amplification and sequencing. organisms growing in extreme environments, we need to develop genetic systems to study biological pro-The rpoB-encoded ␤-subunit of RNA polymerase is highly conserved among prokaryotes (Musser 1995; cesses and to help interpret the information from genome-sequencing projects. But how can we carry out Campbell et al. 2001). Rif r mutants have been analyzed in a number of microorganisms, including several genetic studies in organisms with no characterized genetic systems? One approach is to adapt methods from pathogens (see review by Musser 1995), and the mutations resulting in Rif r have been determined. The mutastudying mutations that have been developed in wellstudied microorganisms and to derive them again in tions cluster in the same region as those for E. coli and mostly affect the corresponding residues. Thus, the the new organism of interest. We have recently completed the development of a system for analyzing base rpoB/Rif r system offers an opportunity for developing a genetic system to analyze mutations even in organisms substitutions in Escherichia coli based on sequencing rpoB mutations that generate the rifampicin-resistant (Rif r

We have applied a genetic system for analyzing mutations in Escherichia coli to Deinococcus radiodurans, an extremeophile with an astonishingly high resistance to UV- and ionizing-radiation-induced mutagenesis. Taking advantage of the conservation of the β-subunit of RNA polymerase among most prokaryotes, we derived again in D. radiodurans the rpoB/Rif r system that we developed in E. coli to monitor base substitutions, defining 33 base change substitutions at 22 different base pairs. We sequenced >250 mutations leading to Rif r in D. radiodurans derived spontaneously in wild-type and uvrD (mismatch-repair-deficient) backgrounds and after treatment with N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and 5-azacytidine (5AZ). The specificities of NTG and 5AZ in D. radiodurans are the same as those found for E. coli and other organisms. There are prominent base substitution hotspots in rpoB in both D. radiodurans and E. coli. In several cases these are at different points in each organism, even though the DNA sequences surrounding the hotspots and their corresponding sites are very similar in both D. radiodurans and E. coli. In one case the hotspots occur at the same site in both organisms.