A Major Role of the RecFOR Pathway in DNA Double-Strand-Break Repair through ESDSA in Deinococcus radiodurans

Abstract

In Deinococcus radiodurans, the extreme resistance to DNA–shattering treatments such as ionizing radiation or desiccation is correlated with its ability to reconstruct a functional genome from hundreds of chromosomal fragments. The rapid reconstitution of an intact genome is thought to occur through an extended synthesis-dependent strand annealing process (ESDSA) followed by DNA recombination. Here, we investigated the role of key components of the RecF pathway in ESDSA in this organism naturally devoid of RecB and RecC proteins. We demonstrate that inactivation of RecJ exonuclease results in cell lethality, indicating that this protein plays a key role in genome maintenance. Cells devoid of RecF, RecO, or RecR proteins also display greatly impaired growth and an important lethal sectoring as bacteria devoid of RecA protein. Other aspects of the phenotype of recFOR knock-out mutants paralleled that of a ΔrecA mutant: ΔrecFOR mutants are extremely radiosensitive and show a slow assembly of radiation-induced chromosomal fragments, not accompanied by DNA synthesis, and reduced DNA degradation. Cells devoid of RecQ, the major helicase implicated in repair through the RecF pathway in E. coli, are resistant to γ-irradiation and have a wild-type DNA repair capacity as also shown for cells devoid of the RecD helicase; in contrast, ΔuvrD mutants show a markedly decreased radioresistance, an increased latent period in the kinetics of DNA double-strand-break repair, and a slow rate of fragment assembly correlated with a slow rate of DNA synthesis. Combining RecQ or RecD deficiency with UvrD deficiency did not significantly accentuate the phenotype of ΔuvrD mutants. In conclusion, RecFOR proteins are essential for DNA double-strand-break repair through ESDSA whereas RecJ protein is essential for cell viability and UvrD helicase might be involved in the processing of double stranded DNA ends and/or in the DNA synthesis step of ESDSA.

DNA polymerases are error-prone at RecA-mediated recombination intermediates

“…Consistent with this view, UvrD-a SF1 type helicase-is implicated in RDR in Deinococcus radiodurans. 39 In yeast, the replicative hexameric MCM helicase is likely to be loaded at D-loops during BIR. 40 However, BIR is only decreased by ~4-5 fold in the absence of MCM activity, suggesting that other potentially nonprocessive helicases may also function in this pathway, which could conceivably reduce the fidelity of RDR.…”

ATP-Binding Cassette Properties of Recombination Mediator Protein RecF

“…Later, it was shown that recF mutants are even more hypersensitive to UV radiation than RecBC, that RecF pathway plays a major role in replication restart under UV damage conditions, and that RecF is involved in DSBs repair in the absence of RecBC and SbcBC (Clark, 1991;Courcelle, 2005;Courcelle et al, 1997;Ivancic-Bace et al, 2003;Kidane et al, 2004;Kusano et al, 1989;Whitby and Lloyd, 1995;Zahradka et al, 2006). Sequencing of new genomes revealed the ubiquitous nature of RecF pathway proteins found in most bacteria (Rocha et al, 2005), including the radiation resistant bacteria Deinococcus radiodurans (Bentchikou et al, 2010;Cox et al, 2010;Chang et al, 2010;Makarova et al, addition to initial loading of RecA, RecOR further stimulate homologous recombination by preventing the dissociation of RecA* filament from ssDNA in E. coli (Bork et al, 2001). Somewhat different properties were reported for Bacillus subtilis RecO, which does not require RecR for initiation of RecA* formation (Manfredi et al, 2008;Manfredi et al, 2010).…”

A Recombination Puzzle Solved: Role for New DNA Repair Systems in Helicobacter pylori Diversity/Persistence

“…Unlike the addA (recB) mutant, the recR and recO mutants did not show significant sensitivity to ionizing radiation (IR) and to mitomycin C [68,71], suggesting that RecRO pathway is not responsible for repairing DNA damage induced by these agents, most likely double strand breaks. This is in contrast to E. coli where the RecFOR pathway sometimes substitutes for the RecBCD pathway and in Deinococcus radiodurance where the RecFOR pathway plays a major role in double strand break repair [98,99]. On the other hand, H. pylori recR and recO mutants were shown to be much more sensitive to oxidative stress and to acid stress than the wild type strain [71], indicating that H. pylori RecRO pathway is involved in repairing DNA damage induced by these stress conditions.…”