TY - JOUR
T1 - RNase HII Saves rnhA Mutant Escherichia coli from R-Loop-Associated Chromosomal Fragmentation
AU - Kouzminova, Elena A.
AU - Kadyrov, Farid F.
AU - Kuzminov, Andrei
N1 - Funding Information:
We would like to thank all members of this laboratory for enthusiastic discussion of our results and Sharik Khan for strains and plasmids. Bill Metcalf (this department) generously helped us with fluorescent microscopy and offered to use his microscope. We are grateful to Bénédicte Michel for her interest in this work and for helpful suggestions to clarify the presentation. This work was supported by Grant No. GM 073115 from the National Institutes of Health . The authors have no conflict of interest to declare. Appendix A
Publisher Copyright:
© 2017 Elsevier Ltd
PY - 2017/9/15
Y1 - 2017/9/15
N2 - 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.
AB - 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.
KW - R-lesions
KW - R-loops
KW - SOS response
KW - double-strand DNA breaks
KW - stable DNA replication
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U2 - 10.1016/j.jmb.2017.08.004
DO - 10.1016/j.jmb.2017.08.004
M3 - Article
C2 - 28821455
AN - SCOPUS:85028356127
SN - 0022-2836
VL - 429
SP - 2873
EP - 2894
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 19
ER -