Efficient, robust, and versatile fluctuation data analysis using MLE MUtation Rate calculator (mlemur)

The fluctuation assay remains an important tool for analyzing the levels of mutagenesis in microbial populations. The mutant counts originating from some average number of mutations are usually assumed to obey the Luria–Delbrück distribution. While several tools for estimating mutation rates are available, they sometimes lack accuracy or versatility under non-standard conditions. In this work, extensions to the Luria–Delbrück protocol to account for phenotypic lag and cellular death with either perfect or partial plating were developed. Hence, the novel MLE MUtation Rate calculator, or mlemur, is the first tool that provides a user-friendly graphical interface allowing the researchers to model their data with consideration for partial plating, differential growth of mutants and non-mutants, phenotypic lag, cellular death, variability of the final number of cells, post-exponential-phase mutations, and the size of the inoculum. Additionally, mlemur allows the users to incorporate most of these special conditions at the same time to obtain highly accurate estimates of mutation rates and P values, confidence intervals for an arbitrary function of data (such as fold), and perform power analysis and sample size determination for the likelihood ratio test. The accuracy of point and interval estimates produced by mlemur against historical and simulated fluctuation experiments are assessed. Both mlemur and the analyses in this work might be of great help when evaluating fluctuation experiments and increase the awareness of the limitations of the widely-used Lea–Coulson formulation of the Luria–Delbrück distribution in the more realistic biological contexts

Strand specificity of ribonucleotide excision repair in Escherichia coli

In Escherichia coli, replication of both strands of genomic DNA is carried out by a single replicase—DNA polymerase III holoenzyme (pol III HE). However, in certain genetic backgrounds, the low-fidelity TLS polymerase, DNA polymerase V (pol V) gains access to undamaged genomic DNA where it promotes elevated levels of spontaneous mutagenesis preferentially on the lagging strand. We employed active site mutants of pol III (pol IIIα_S759N) and pol V (pol V_Y11A) to analyze ribonucleotide incorporation and removal from the E. coli chromosome on a genome-wide scale under conditions of normal replication, as well as SOS induction. Using a variety of methods tuned to the specific properties of these polymerases (analysis of lacI mutational spectra, lacZ reversion assay, HydEn-seq, alkaline gel electrophoresis), we present evidence that repair of ribonucleotides from both DNA strands in E. coli is unequal. While RNase HII plays a primary role in leading-strand Ribonucleotide Excision Repair (RER), the lagging strand is subject to other repair systems (RNase HI and under conditions of SOS activation also Nucleotide Excision Repair). Importantly, we suggest that RNase HI activity can also influence the repair of single ribonucleotides incorporated by the replicase pol III HE into the lagging strand

Novel Escherichia coli active site dnaE alleles with altered base and sugar selectivity

The Escherichia coli dnaE gene encodes the α‐catalytic subunit (pol IIIα) of DNA polymerase III, the cell’s main replicase. Like all high‐fidelity DNA polymerases, pol III possesses stringent base and sugar discrimination. The latter is mediated by a so‐called “steric gate” residue in the active site of the polymerase that physically clashes with the 2′‐OH of an incoming ribonucleotide. Our structural modeling data suggest that H760 is the steric gate residue in E.coli pol IIIα. To understand how H760 and the adjacent S759 residue help maintain genome stability, we generated DNA fragments in which the codons for H760 or S759 were systematically changed to the other nineteen naturally occurring amino acids and attempted to clone them into a plasmid expressing pol III core (α‐θ‐ε subunits). Of the possible 38 mutants, only nine were successfully sub‐cloned: three with substitutions at H760 and 6 with substitutions at S759. Three of the plasmid‐encoded alleles, S759C, S759N, and S759T, exhibited mild to moderate mutator activity and were moved onto the chromosome for further characterization. These studies revealed altered phenotypes regarding deoxyribonucleotide base selectivity and ribonucleotide discrimination. We believe that these are the first dnaE mutants with such phenotypes to be reported in the literature