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 - 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
UR - http://www.scopus.com/inward/record.url?scp=85028356127&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85028356127&partnerID=8YFLogxK
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 -