RNase HII Saves rnhA Mutant Escherichia coli from R-Loop-Associated Chromosomal Fragmentation

Research output: Contribution to journalArticlepeer-review

Abstract

The rnhAB mutant Escherichia coli, deficient in two RNase H enzymes that remove both R-loops and incorporated ribonucleotides (rNs) from DNA, grow slowly, suggesting accumulation of rN-containing DNA lesions (R-lesions). We report that the rnhAB mutants have reduced viability, form filaments with abnormal nucleoids, induce SOS, and fragment their chromosome, revealing replication and/or segregation stress. R-loops are known to interfere with replication forks, and sensitivity of the double rnhAB mutants to translation inhibition points to R-loops as precursors for R-lesions. However, the strict specificity of bacterial RNase HII for RNA–DNA junctions indicates that R-lesions have rNs integrated into DNA. Indeed, instead of relieving problems of rnhAB mutants, transient inhibition of replication from oriC kills them, suggesting that oriC-initiated replication removes R-loops instead of compounding them to R-lesions. Yet, replication from an R-loop-initiating plasmid origin kills the double rnhAB mutant, revealing generation of R-lesions by R-loop-primed DNA synthesis. These R-lesions could be R-tracts, contiguous runs of ≥ 4 RNA nucleotides within DNA strand and the only common substrate between the two bacterial RNase H enzymes. However, a plasmid relaxation test failed to detect R-tracts in DNA of the rnhAB mutants, although it readily detected R-patches (runs of 1–3 rNs). Instead, we detected R-gaps, single-strand gaps containing rNs, in the chromosomal DNA of the rnhAB mutant. Therefore, we propose that RNase H-deficient mutants convert some R-loops into R-tracts, which progress into R-gaps and then to double-strand breaks—explaining why R-tracts do not accumulate in RNase H-deficient cells, while double-strand breaks do.

Original languageEnglish (US)
Pages (from-to)2873-2894
Number of pages22
JournalJournal of Molecular Biology
Volume429
Issue number19
DOIs
StatePublished - Sep 15 2017

Keywords

  • R-lesions
  • R-loops
  • SOS response
  • double-strand DNA breaks
  • stable DNA replication

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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