Jayaraman Gowrishankar

The essential homotetrameric endoribonuclease RNase E of Escherichia coli participates in global RNA turnover as well as stable RNA maturation. The protomer’s N-terminal half (residues 1–529) bears the catalytic, allosteric, and tetramerization domains, including the active site residues D303 and D346. The C-terminal half (CTH, residues 530–1061) is dispensable for viability. We have previously described a phenomenon of recessive resurrection in RNase E that requires the CTH, wherein the wild-type homotetramer apparently displays nearly identical activity in vivo as a heterotetramer comprising three catalytically dead subunits (with D303A or D346A substitutions) and one wild-type subunit. Here, we show that recessive resurrection is exhibited even in dimeric RNase E with the CTH, and that it is largely dependent on the presence of a membrane-targeting-sequence motif (residues 565–582). A single F575E substitution also impaired recessive resurrection, whereas other CTH motifs (such as those for binding of RNA or of partner proteins) were dispensable. The phenomenon was independent of RNA 5′-monophosphate sensing by the enzyme. We propose that membrane-anchoring of RNase E renders it processive for endoribonucleolytic action, and that recessive resurrection and dominant negativity associated with mutant protomers are mutually exclusive manifestations of, respectively, processive and distributive catalytic mechanisms in a homo-oligomeric enzyme.

In many bacteria, the essential factors Rho and NusG mediate termination of synthesis of nascent transcripts (including antisense RNAs) that are not being simultaneously translated. It has been proposed that in Rho’s absence toxic RNA–DNA hybrids (R-loops) may be generated from nascent untranslated transcripts, and genome-wide mapping studies in Escherichia coli have identified putative loci of R-loop formation from more than 100 endogenous antisense transcripts that are synthesized only in a Rho-deficient strain. Here we provide evidence that engineered expression in wild-type E. coli of several such individual antisense regions on a plasmid or the chromosome generates R-loops that, in an RNase H-modulated manner, serve to disrupt genome integrity. Rho inhibition was associated with increased prevalence of antisense R-loops also in Xanthomonas oryzae pv. oryzae and Caulobacter crescentus. Our results confirm the essential role of Rho in several bacterial genera for prevention of toxic R-loops from pervasive yet cryptic endogenous antisense transcripts. Engineered antisense R-looped regions may be useful for studies on both site-specific impediments to bacterial chromosomal replication and the mechanisms of their resolution.

In many bacteria, the essential factors Rho and NusG mediate termination of synthesis of nascent transcripts (including antisense RNAs) which are not being simultaneously translated. It has been proposed that in Rho’s absence toxic RNA-DNA hybrids (R-loops) may be generated from nascent untranslated transcripts; and genome-wide mapping studies in Escherichia coli have identified putative loci of R-loop formation from more than 100 endogenous antisense transcripts that are synthesized only in a Rho-deficient strain. Here we provide evidence that engineered expression in wild-type E. coli of several such individual antisense regions on a plasmid or the chromosome generates R-loops that, in an RNase H-modulated manner, serve to disrupt genome integrity. Rho inhibition was associated with increased prevalence of antisense R-loops also in Xanthomonas oryzae pv. oryzae and Caulobacter crescentus . Our results confirm the essential role of Rho in several bacterial genera for prevention of toxic R-loops from pervasive yet cryptic endogenous antisense transcripts. Engineered antisense R-looped regions may be useful for studies on both site-specific impediments to bacterial chromosomal replication and the mechanisms of their resolution.

The essential homo-tetrameric endoribonuclease RNase E of Escherichia coli participates in global RNA turnover as well as stable RNA maturation. The protomer’s N-terminal half (residues 1-529) bears the catalytic, allosteric and tetramerization domains, including the critical active site residues D303 and D346. The C-terminal half (CTH, residues 530-1061) is dispensable for viability. We have previously described a phenomenon of recessive resurrection in RNase E that requires the CTH, wherein the wild-type homo-tetramer apparently displays nearly identical activity in vivo as a hetero-tetramer comprised of three catalytically dead subunits (with D303A/D346A substitutions) and one wild-type subunit. Here we show that recessive resurrection is exhibited even in dimeric RNase E with the CTH, and that it is largely dependent on presence of the membrane-targeting-sequence motif (residues 565-582). A single F575E substitution also impaired recessive resurrection, whereas other CTH motifs (such as those for binding of RNA or of partner proteins) were dispensable. The phenomenon was independent of RNA 5’-monophosphate sensing by the enzyme. We propose that membrane-anchoring of RNase E renders it processive for endoribonucleolytic action, and that recessive resurrection and dominant negativity associated with mutant protomers are mutually exclusive manifestations of, respectively, processive and distributive catalytic mechanisms in a homo-oligomeric enzyme.

Replication of the circular bacterial chromosome is initiated from a locus oriC with the aid of an essential protein DnaA. One approach to identify factors acting to prevent aberrant oriC-independent replication initiation in Escherichia coli has been that to obtain mutants which survive loss of DnaA. Here, we show that a ΔrecD mutation, associated with attenuation of RecBCD’s DNA double strand end-resection activity, provokes abnormal replication and rescues ΔdnaA lethality in two situations: (i) in absence of 5′-3′ single-strand DNA exonuclease RecJ, or (ii) when multiple two-ended DNA double strand breaks (DSBs) are generated either by I-SceI endonucleolytic cleavages or by radiomimetic agents phleomycin or bleomycin. One-ended DSBs in the ΔrecD mutant did not rescue ΔdnaA lethality. With two-ended DSBs in the ΔrecD strain, ΔdnaA viability was retained even after linearization of the chromosome. Data from genome-wide DNA copy number determinations in ΔdnaA-rescued cells lead us to propose a model that nuclease-mediated DNA resection activity of RecBCD is critical for prevention of a σ-mode of rolling-circle over-replication when convergent replication forks merge and fuse, as may be expected to occur during normal replication at the chromosomal terminus region or during repair of two-ended DSBs following ‘ends-in’ replication.

Replication of the circular bacterial chromosome is initiated from a locus oriC with the aid of an essential protein DnaA. One approach to identify factors acting to prevent aberrant oriC- independent replication initiation in Escherichia coli has been that to obtain mutants which survive loss of DnaA. Here we show that a Δ recD mutation, associated with attenuation of RecBCD’s DNA double strand end-resection activity, provokes abnormal replication and rescues Δ dnaA lethality in two situations: (i) in absence of 5’-3’ single-strand DNA exonuclease RecJ, or (ii) when multiple two-ended DNA double strand breaks (DSBs) are generated either by I-SceI endonucleolytic cleavages or by radiomimetic agents phleomycin or bleomycin. One-ended DSBs in the Δ recD mutant did not rescue Δ dnaA lethality. With two-ended DSBs in the Δ recD strain, Δ dnaA viability was retained even after linearization of the chromosome. Data from genome-wide DNA copy number determinations in Δ dnaA -rescued cells lead us to propose a model that nuclease-mediated DNA resection activity of RecBCD is critical for prevention of a σ-mode of rolling-circle over-replication when convergent replication forks merge and fuse, as may be expected to occur during normal replication at the chromosomal terminus region or during repair of two-ended DSBs following “ends-in” replication.

Double-strand breaks (DSBs) in DNA are major threats to genome integrity. In Escherichia coli , DSBs are repaired by RecA- and RecBCD-mediated homologous recombination (HR).

The process of homologous recombination (HR) is important for maintenance of genome integrity in all cells. In Escherichia coli , the RecA protein is a critical participant in HR, which acts at a step common to and downstream of two HR pathways mediated by the RecBCD and RecFOR proteins, respectively. In this study, an isoform (IF2-1) of the translation initiation factor IF2 has been identified as a novel facilitator of RecA’s function in vivo during HR.

In Escherichia coli , three isoforms of the essential translation initiation factor IF2 (IF2-1, IF2-2, and IF2-3) are generated from separate in-frame initiation codons in infB . The isoforms have earlier been suggested to additionally participate in DNA damage repair and replication restart. It is also known that the proteins RecA and RecBCD are needed for repair of DNA double-strand breaks (DSBs) in E. coli . Here we show that strains lacking IF2-1 are profoundly sensitive to two-ended DSBs in DNA generated by radiomimetic agents phleomycin or bleomycin, or by endonuclease I-SceI. However, these strains remained tolerant to other DSB-generating genotoxic agents or perturbations to which recA and recBC mutants remained sensitive, such as to mitomycin C, type-2 DNA topoisomerase inhibitors, or DSB caused by palindrome cleavage behind a replication fork. Data from genome-wide copy number analyses following I-SceI cleavage at a single chromosomal locus suggested that, in a strain lacking IF2-1, the magnitude of break induced replication through replication restart mechanisms is largely preserved but the extent of DNA resection around the DSB site is reduced. We propose that in absence of IF2-1 it is the annealing of a RecA nucleoprotein filament to its homologous target that is weakened, which in turn leads to a specific failure in assembly of Ter-to-oriC directed replisomes needed for consummation of two-ended DSB repair. Importance: Double-strand breaks (DSBs) in DNA are major threats to genome integrity. In Escherichia coli , DSBs are repaired by Rec- and RecBCD-mediated homologous recombination (HR). This study demonstrates a critical role for an isoform (IF2-1) of the translation initiation factor IF2 in the repair of two-ended DSBs in E. coli (that can be generated by ionizing radiation, certain DNA-damaging chemicals, or endonuclease action). It is proposed that IF2-1 acts to facilitate the function of RecA in the annealing between a pair of DNA molecules during HR.

Topoisomerase I (Topo I) of Escherichia coli, encoded by topA, acts to relax negative supercoils in DNA. Topo I deficiency results in hypernegative supercoiling, formation of transcription-associated RNA-DNA hybrids (R-loops), and DnaA- and oriC-independent constitutive stable DNA replication (cSDR), but some uncertainty persists as to whether topA is essential for viability in E. coli and related enterobacteria. Here we show that several topA alleles, including ΔtopA, confer lethality in derivatives of wild-type E. coli strain MG1655. Viability in absence of Topo I was restored with two perturbations, neither of which reversed the hypernegative supercoiling phenotype: (i) in a reduced-genome strain MDS42, or (ii) by an RNA polymerase (RNAP) mutation rpoB*35 that has been reported to alleviate the deleterious consequences of RNAP backtracking and transcription-replication conflicts. Four phenotypes related to cSDR were identified for topA mutants: (i) One of the topA alleles rescued ΔdnaA lethality; (ii) in dnaA+ derivatives, Topo I deficiency generated a characteristic copy number peak in the terminus region of the chromosome; (iii) topA was synthetically lethal with rnhA (encoding RNase HI, whose deficiency also confers cSDR); and (iv) topA rnhA synthetic lethality was itself rescued by ΔdnaA. We propose that the terminal lethal consequence of hypernegative DNA supercoiling in E. coli topA mutants is RNAP backtracking during transcription elongation and associated R-loop formation, which in turn lead to transcription-replication conflicts and to cSDR. Importance In all life forms, double helical DNA exists in a topologically supercoiled state. The enzymes DNA gyrase and topoisomerase I act, respectively, to introduce and to relax negative DNA supercoils in Escherichia coli. That gyrase deficiency leads to bacterial death is well established, but the essentiality of topoisomerase I for viability has been less certain. This study confirms that topoisomerase I is essential for E. coli viability, and suggests that in its absence aberrant chromosomal DNA replication and excessive transcription-replication conflicts occur that are responsible for lethality.