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

Abstract

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.

Escherichia coli DNA replication: the old model organism still holds many surprises

“…However, genetic studies reveal that Pol III mutations leading to a mutator phenotype are sometimes located in amino acids that do not make direct contact with DNA or the incoming nucleotide. For example, mutations in Pol III Serine 759 are thought to cause impaired closing of the fingers domain over the palm domain during catalysis, which might contribute to increased mutagenesis observed in vivo for example due to improper geometry of the active site in the closed conformation (Parasuram et al 2018 , Vaisman et al 2021 ). Apart from that, the propensity of DNA polymerases to mispair deoxyribonucleotides also depends on the sequence context and their capability to extend the mismatch.…”