Non-canonical processing of DNA photodimers with Bacillus subtilis UV-endonuclease YwjD, 5 '- 3 ' exonuclease YpcP and low-fidelity DNA polymerases YqjH and YqjW

摘要

It has been shown that mutation frequency decline (Mfd) and nucleotide excision repair (NER) deficiencies promote UV-induced mutagenesis in B. subtilis sporangia. As replication is halted in sporulating B. subtilis cells, in this report, we investigated if this response may result from an error-prone repair event involving the UV-endonuclease YwjD and low fidelity (LF) DNA synthesis. Accordingly, disruption of YwjD generated B. subtilis sporangia that were more susceptible to UV-C radiation than sporangia of the WT strain and such susceptibility increased even more after the single or simultaneous inactivation of the LF DNA polymerases YqjH and YqjW. To further explore this concept, functional His(6)-tagged YwjD and Y-DNA polymerases YqjH and YqjW were produced and purified to homogeneity. In vitro repair assays showed that YwjD hydrolyzed the phosphodiester bond immediately located 5'-end of a ds-DNA substrate bearing either, cyclobutane pyrimidine dimers (CPD), 6-4 photoproducts (6-4 PD) or Dewar isomers (DWI). Notably, the 6-4 PD and DWI but not the CPDs repair intermediaries of YwjD were efficiently processed by the LF polymerase YqjH suggesting that an additional 5'- 3' exonuclease event was necessary to process PD. Accordingly, the LF polymerase YqjW efficiently processed the incision-excision repair products derived from YwjD and exonuclease YpcP attack over CPD-containing DNA. In summary, our results unveiled a novel non-canonical repair pathway that employs YwjD to incise PD-containing DNA and low fidelity synthesis contributing thus to mutagenesis, survival and spore morphogenesis in B. subtilis.