Topoisomerase I Essentiality, DnaA-Independent Chromosomal Replication, and Transcription-Replication Conflict in Escherichia coli

Abstract

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 Δ topA , 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 Δ dnaA 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 Δ dnaA . 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.

Role for DNA double strand end-resection activity of RecBCD in control of aberrant chromosomal replication initiation in Escherichia coli

“…Leach and coworkers had shown excessive DNA synthesis in the vicinity of a site-specific chromosomal DSB in a ΔrecD mutant (49). Furthermore, copy number determinations by WGS in ΔrecD strains (48,49) have revealed a characteristic "mid-terminus peak" that is most commonly associated with mutants exhibiting cSDR (2,(26)(27)(28)(29)(30).…”

“…Dispensability of rpoB*35 for cSDR-mediated ∆dnaA viability in Δrrn strains. In all situations where cSDR has been shown to confer viability in absence of DnaA function, the rpoB*35 mutation (along with ∆tus) is either required for, or enhances the degree of, lethality rescue (25,(27)(28)(29)(30). The model is that oriC-independent replication encounters substantial conflicts with transcription which thereby limit its ability to cover the entire chromosome (2), and that rpoB*35 facilitates this process by mitigating the deleterious consequences of transcription-replication conflict (31)(32)(33)(34).…”

“…Three approaches were used in this work to study occurrence and features of cSDR in different strains. As described above, a shelter plasmid-based ‘blue-white screening’ strategy was used initially to identify conditions that serve to rescue Δ dnaA lethality (28, 29). In a second approach based on flow cytometry, the extent of continued DNA synthesis following addition of a protein synthesis inhibitor spectinomycin was investigated (29, 45).…”

“…Blue-white assay for determining viability of ΔdnaA derivatives. The 'blue-white screening' strategy used to identify conditions that render a ∆dnaA mutant viable was as earlier described (28,29). The parental strain is ∆dnaA (and ∆lacZ) on the chromosome and carries a single-copy-number Tp R dnaA + lacZ + shelter plasmid pHYD2388 whose equi-partitioning to daughter cells during cell division is inefficient; thus, typically around 5 to 20% of cells in a culture (grown in absence of Tp) are plasmid-free.…”

“…Such mutants are said to exhibit constitutive SDR [cSDR (24)]. The mutations that have been so identified include those abrogating the functions of RNase HI (24–26); RecG helicase (25, 27); topoisomerase I (28); Dam DNA methylase (29); or different combinations of three redundant 3’ ss-DNA exonucleases I, VII, and SbcCD, and a 5’ ss-DNA exonuclease RecJ (30). In all of these cases, two additional mutations (Δ tus and rpoB*35 ) are also required for (or, in case of RNase HI deficiency, facilitate) DnaA-independent viability (25, 27–30); the two mutations presumably aid in the progression of oriC -independent replication completely around the circular chromosome, the former by disrupting the polar replication barriers at Ter sequences (4, 5) and the latter by resolving head-on collisions of transcription complexes with the replisome (31–34).…”