RecG Protein and Single-Strand DNA Exonucleases Avoid Cell Lethality Associated With PriA Helicase Activity inEscherichia coli

Abstract

Replication of the Escherichia coli chromosome usually initiates at a single origin (oriC) under control of DnaA. Two forks are established and move away in opposite directions. Replication is completed when these meet in a broadly defined terminus area half way around the circular chromosome. RecG appears to consolidate this arrangement by unwinding D-loops and R-loops that PriA might otherwise exploit to initiate replication at other sites. It has been suggested that without RecG such replication generates 39 flaps as the additional forks collide and displace nascent leading strands, providing yet more potential targets for PriA. Here we show that, to stay alive, cells must have either RecG or a 39 single-stranded DNA (ssDNA) exonuclease, which can be exonuclease I, exonuclease VII, or SbcCD. Cells lacking all three nucleases are inviable without RecG. They also need RecA recombinase and a Holliday junction resolvase to survive rapid growth, but SOS induction, although elevated, is not required. Additional requirements for Rep and UvrD are identified and linked with defects in DNA mismatch repair and with the ability to cope with conflicts between replication and transcription, respectively. Eliminating PriA helicase activity removes the requirement for RecG. The data are consistent with RecG and ssDNA exonucleases acting to limit PriA-mediated re-replication of the chromosome and the consequent generation of linear DNA branches that provoke recombination and delay chromosome segregation.

The Nucleotide Excision Repair System of Borrelia burgdorferi Is the Sole Pathway Involved in Repair of DNA Damage by UV Light

“…These results suggest that, even in the presence of a functional NER system, recombinational repair (recA, ruvABC, recG, and recBCD), transcriptioncoupled repair (mfd), and repair of arrested replication forks (recG, priA) play a significant role in the repair of UV damage in N. gonorrhoeae, but not in B. burgdorferi. No orthologs of sbcCD (70) are found in the sequenced genome of N. gonorrhoeae, but their disruption did not affect B. burgdorferi survival to UV damage. Other recent studies have also reported that B. burgdorferi recA and ruvA are dispensable for repairing UV-induced DNA damage (39,40).…”

Homologous Recombination—Experimental Systems, Analysis, and Significance

“…The prediction is that, if performed in the recBC sbcA or recBC sbcBC backgrounds, such a screen should yield all other known rec mutants, as well as (hopefully) some still unknown ones. As an approach, identification of synthetic lethal mutant combinations shows its power in unraveling complicated pathways of the DNA metabolism around recombinational repair (270, 357), although care should be exercised with interpretation of quadruple and quintuple mutants, as the comfort zone of typical human thinking is limited to three dimensions.…”

Exonuclease VII is involved in “reckless” DNA degradation in UV-irradiated Escherichia coli

“…Also, our present and previous results suggest that the nuclease action on 3 -ssDNA is particularly important as a stimulator of ExoV activity during "reckless" DNA degradation. Such a notion is corroborated by the accumulated genetic data indicating that ExoI, ExoVII and SbcCD play partially redundant roles in removing 3 -ssDNA tails [27,30,31,[49][50][51]. ExoVII possesses both 3 -5 and 5 -3 polarities on ssDNA [45], which makes it possible that either of its activities is involved in the regulation of "reckless" DNA degradation.…”

“…Several genetic studies suggest that ExoVII plays an overlapping role with ExoI and/or SbcCD in removing 3 -ended single-stranded tails from DNA duplexes [29,31,[49][50][51]. Hence, these results indicate that ExoVII activity might also influence the extent of "reckless" degradation in recA cells.…”