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

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

Mécanismes de réparation des cassures double crin de l ADN chez la bactérie radiorésistante deinococcus radiodurans

La bactérie Deinococcus radiodurans présente une résistance exceptionnelle aux agents qui endommagent l ADN et en particulier aux radiations ionisantes. Cette résistance est liée à sa capacité de reconstituer rapidement un génome intact à partir de centaines de fragments. Cette reconstitution fait intervenir un mécanisme appelé Extended Synthesis-Dependant Strand Annealing (ESDSA) dépendant d une synthèse active d ADN et la recombinaison homologue. Peu de choses sont connues sur les facteurs nécessaires à la formation des extrémités 3 simple brin qui vont servir d amorce pour la synthèse massive d ADN. D. radiodurans est dépourvu des proteines RecB et RecC mais exprme SbcC et SbcD, homologues des protéines eucaryotes Rad50 et Mre11, PolXDr, une polymérase de la famille X possédant une activité 3 -5 exonucléase et RecJ, RecQ, RecF, RecO et RecR, facteurs clé de la voie RecF. Nous avons montré que le complexe SbcCD et la polymérase PolXDr ont des activités partiellement chevauchantes et participent à la réparation de lésions complexes et difficiles à réparer. L exonucléase RecJ, en association avec l hélicase UvrD, pourrait engendrer des extrémités 3 simple brin sur lesquelles le complexe RecFOR stimulerait le chargement de RecA pour initier l ESDSA. De plus, nous avons montré, en utilisant un essai de recombinaison que j ai mis au point, que la recombinaison spontanée entre des séquences répétées distantes de 1500 pb est indépendante de RecA et de RecFOR suggérant un mécanisme de glissement de polymérase, stimulée par l absence d UvrD, et induite par exposition aux radiations ionisantes.The Deinococcus radiodurans bacterium is extremely resistant to treatments causing extensive DNA double strand breaks including ionizing radiations. This resistance is linked to the ability of D. radiodurans to reconstruct a functional genome from hundreds of chromosomal fragments. This reconstitution is dependent on extended synthesis-dependent strand annealing (ESDSA) and homologous recombination. Little is known about the cellular factors required for the formation of single-stranded 3 overhangs which, through RecA mediated strand invasion, prime DNA synthesis. D. radiodurans is naturally devoid of RecB and RecC proteins but possesses SbcC and SbcD, the homologues of the eukaryotic Rad50 and Mre11 proteins, PolXDr, a polymerase of the X family that possesses a structure-modulated 3 -5 exonuclease activity and RecJ, RecQ, RecF, RecO and RecR proteins, the key components of the RecF pathway. We have shown that the SbcCD complex and the PolXDr polymerase possess partially redondant activities and are part of a back-up repair system acting to rescue cells containing DNA lesions that are excessively numerous or difficult to repair. The RecJ exonuclease, in conjunction with the UvrD helicase, may generate the single-stranded tails on which RecFOR stimulates RecA loading. Moreover, using a recombination assay, we found that the recombination between direct repeats separated by a 1500 bp spacer is RecA and RecFOR independent suggesting the presence in D. radiodurans of an efficient replication slippage mechanism, stimulated by the absence of UvrD and induced by ionizing radiations.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Roles of PprA, IrrE, and RecA in the resistance of Deinococcus radiodurans to germicidal and environmentally relevant UV radiation

To study the role of different DNA repair genes in the resistance of Deinococcus radiodurans to mono- and polychromatic UV radiation, wild-type strain and knockout mutants in RecA, PprA, and IrrE of D. radiodurans were irradiated with UV-C (254 nm), UV-(A + B) (280–400 nm) and UV-A (315–400 nm) radiation, and survival was monitored. The strain deficient in recA was highly sensitive to UV-C radiation compared to the wild-type, but showed no loss of resistance against irradiation with UV-(A + B) and UV-A, while pprA and irrE-deficient strains exhibited elevated sensitivity to UV-A and UV-(A + B) radiation. These results suggest that the repair of DNA double-strand breaks is essential after treatment with highly energetic UV-C radiation, whereas protection from oxidative stress may play a greater role in resistance to environmentally relevant UV radiation

Additive Effects of SbcCD and PolX Deficiencies in the In Vivo Repair of DNA Double-Strand Breaks in Deinococcus radiodurans▿ †

Orthologs of proteins SbcD (Mre11) and SbcC (Rad50) exist in all kingdoms of life and are involved in a wide variety of DNA repair and maintenance functions, including homologous recombination and nonhomologous end joining. Here, we have inactivated the sbcC and/or sbcD genes of Deinococcus radiodurans, a highly radioresistant bacterium able to mend hundreds of radiation-induced DNA double-strand breaks (DSB). Mutants devoid of the SbcC and/or SbcD proteins displayed reduced survival and presented a delay in kinetics of DSB repair and cell division following γ-irradiation. It has been recently reported that D. radiodurans DNA polymerase X (PolX) possesses a structure-modulated 3′-to-5′ exonuclease activity reminiscent of specific nuclease activities displayed by the SbcCD complex from Escherichia coli. We constructed a double mutant devoid of SbcCD and PolX proteins. The double-mutant ΔsbcCD ΔpolXDr (where Dr indicates D. radiodurans) bacteria are much more sensitive to γ-irradiation than the single mutants, suggesting that the deinococcal SbcCD and PolX proteins may play important complementary roles in processing damaged DNA ends. We propose that they are part of a backup repair system acting to rescue cells containing DNA lesions that are excessively numerous or difficult to repair

Are the SSB-Interacting Proteins RecO, RecG, PriA and the DnaB-Interacting Protein Rep Bound to Progressing Replication Forks in Escherichia coli?

International audienceIn all organisms several enzymes that are needed upon replication impediment are targeted to replication forks by interaction with a replication protein. In most cases these proteins interact with the polymerase clamp or with single-stranded DNA binding proteins (SSB). In Escherichia coli an accessory replicative helicase was also shown to interact with the DnaB replicative helicase. Here we have used cytological observation of Venus fluorescent fusion proteins expressed from their endogenous loci in live E. coli cells to determine whether DNA repair and replication restart proteins that interact with a replication protein travel with replication forks. A custom-made microscope that detects active replisome molecules provided that they are present in at least three copies was used. Neither the recombination proteins RecO and RecG, nor the replication accessory helicase Rep are detected specifically in replicating cells in our assay, indicating that either they are not present at progressing replication forks or they are present in less than three copies. The Venus-PriA fusion protein formed foci even in the absence of replication forks, which prevented us from reaching a conclusion

Neuro-Otology: Problems of Dizziness, Balance and Hearing

© 2016 John Wiley & Sons, Ltd. This chapter expalins basic concepts of dizziness and vertigo, and discusses neuro-otological assessment for dizziness and vertigo. Dizziness is a substantial cause of morbidity, loss of time from work, repeated medical attendances and costly investigation - one study showed that on average more than four physicians were visited before precise diagnosis of a vestibular problem. The chapter presents the vestibular investigations, clinical disorders, and management of vestibular disorders. Direct observation of eye movements is useful, while recording techniques such as electronystagmography, video-oculography (VOG) and spiral coil recordings allow detailed evaluation and provide a permanent record for comparative purposes. Acute unilateral loss of vestibular function gives rise to symptoms of vertigo, nausea, vomiting, sweating, pallor and diarrhea. The chapter also discusses anatomy, physiology, definitions, introduction and basic concepts for hearing disorders. It describes the clinical examination of the ear and hearing, audiological investigations, auditory processing disorders, management of auditory disorders

Bacterial strains and plasmids.

<p>^(a) 5’<i>tetA</i> and the 3’<i>tetA</i> contain the 1–779 and the 342–1190 part of the <i>tetA</i> coding region, respectively. They contain a 342–779 overlapping region. The positions are numbered relatively to the first base of the initiation codon of <i>tetA</i>.</p><p>^(b) The positions of the mutations in the 5’<i>tetA</i> fragment are numbered relatively to the first base of the initiation codon of <i>tetA</i>.</p><p>Bacterial strains and plasmids.</p

Recombination between chromosomal and plasmid DNA is RecA- and RecF-dependent.

<p><b>A.</b> Schematic representation of the recombination assay between chromosomal and plasmid DNA. The 5’<i>tetA</i> and the 3’<i>tetA</i> regions of the <i>tetA</i> gene, containing 438 bp repeats, were introduced into the chromosomal dispensable <i>amyE</i> gene and into the p11554 plasmid giving rise to plasmid p15002, respectively. One crossing over between the two 438 bp repeated sequences (black boxes) leads to the reconstitution of a functional <i>tetA</i> gene and the integration of the plasmid into chromosomal DNA. <b>B.</b> Medians of [Tet^R] frequencies in WT (GY15147), Δ<i>recA</i> (GY15158), Δ<i>recF</i> (GY15160), Δ<i>radA</i> (GY15149), and Δ<i>uvrD</i> (GY15156) bacteria, all containing the p15002 plasmid, are calculated from at least 3 independent values and represented by Tukey boxplots. Outliers are represented by open circles. The small arrows attached to the horizontal line representing the upper limit of detectable [Tet^R] frequencies indicate that [Tet^R] frequencies were < 5 10⁻⁷ for Δ<i>recA</i> and Δ<i>recF</i> bacteria.</p

Characterization of the Radiation Desiccation Response Regulon of the Radioresistant Bacterium Deinococcus radiodurans by Integrative Genomic Analyses

International audienceNumerous genes are overexpressed in the radioresistant bacterium Deinococcus radiodurans after exposure to radiation or prolonged desiccation. It was shown that the DdrO and IrrE proteins play a major role in regulating the expression of approximately twenty genes. The transcriptional repressor DdrO blocks the expression of these genes under normal growth conditions. After exposure to genotoxic agents, the IrrE metalloprotease cleaves DdrO and relieves gene repression. At present, many questions remain, such as the number of genes regulated by DdrO. Here, we present the first ChIP-seq analysis performed at the genome level in Deinococcus species coupled with RNA-seq, which was achieved in the presence or not of DdrO. We also resequenced our laboratory stock strain of D. radiodurans R1 ATCC 13939 to obtain an accurate reference for read alignments and gene expression quantifications. We highlighted genes that are directly under the control of this transcriptional repressor and showed that the DdrO regulon in D. radiodurans includes numerous other genes than those previously described, including DNA and RNA metabolism proteins. These results thus pave the way to better understand the radioresistance pathways encoded by this bacterium and to compare the stress-induced responses mediated by this pair of proteins in diverse bacteria

Impaired growth and stationary-phase lethality of recombination-deficient mutant cells.

<p>GY9613 (WT) (black diamonds), GY13915 (Δ<i>ddrB</i>) (grey squares), GY15125 (Δ<i>recO</i>) (green triangles), GY12968 (Δ<i>recA</i>) (blue squares), GY16626 (Δ<i>ddrB</i> Δ<i>recA</i>) (red squares and interrupted lines), GY16636 (Δ<i>ddrB</i> Δ<i>recO</i>) (orange circles) were grown from independent colonies at 30°C to an OD_650nm = 0.1 (time 0 of the growth curves). <b>A.</b> OD_650nm as a function of time. <b>B.</b> Colony forming units as a function of time. <b>C.</b> Colony forming units as a function of OD_650nm.</p