GSE158570
Genome-wide tracking of Escherichia coli DNA polymerase V to the lagging strand during the SOS response
Ribonucleotides are frequently incorporated into DNA and can be used as a marker of DNA replication enzymology. To investigate on a genome-wide scale, how E. coli pol V accesses undamaged chromosomal DNA during the SOS response, we mapped the location of ribonucleotides incorporated by steric gate variants of pol V across the entire E. coli genome. To do so, we used strains that are deficient in ribonucleotide excision repair (DrnhB), constitutively express all SOS-regulated genes [lexA(Def)] and constitutively “activated” RecA* (recA730).  The strains also harbor two steric gate variants of E. coli pol V (Y11A or F10L), or a homolog of pol V, (pol VR391-Y13A). Ribonucleotides are frequently incorporated by the pol V-Y11A and pol VR391-Y13A variants, with a preference to the lagging strand.  In contrast, the pol V-F10L variant incorporates less ribonucleotides and no strand preference was observed. Sharp transitions in strand specificity are observed at replication origin (oriC), while a gradient is observed at the termination region. To activate RecA* in a recA+ strain, we treated the strains with ciprofloxacin and genome-wide mapped the location of the incorporated ribonucleotides. Again, the polV-Y11A steric gate variant, exhibited a lagging strand preference. Our data is consistent with a specific role for pol V in lagging strand DNA synthesis across the entire E. coli genome during the SOS response. Overall design: Mapping ribonucleotides in DNA from E. coli  that induced SOS-response in combination with various steric gate variants of polV that allow more ribonucleotides to be incororated with HydEn-seq (5´end sequencing of genomic fragments generated by alkaline hydrolysis).