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 III alpha_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.

Automated summary

In Escherichia coli, replication of genomic DNA is mainly carried out by DNA polymerase III holoenzyme (pol III HE). However, in certain genetic backgrounds, DNA polymerase V (pol V) can access undamaged genomic DNA and cause increased spontaneous mutagenesis on the lagging strand. This study investigates the repair of ribonucleotides on both DNA strands in E. coli using active site mutants of pol III and pol V. The findings suggest unequal repair of ribonucleotides, with different repair systems involved in the leading and lagging strands.