RecBC enzyme overproduction affects UV and gamma radiation survival of Deinococcus radiodurans

Khairnar, Nivedita P.; Kamble, Vidya A.; Misra, Hari S.

doi:10.1016/j.dnarep.2007.07.007

Cited by 43 publications

(40 citation statements)

References 43 publications

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“…This suggested that UVC tolerance in this bacterium may be supported by a mechanism that does not require PQQ. Previous findings have also shown that ␥-radiation-sensitive derivatives of D. radiodurans R1 are not always sensitive to UVC radiation (19,34). E. coli does not harbor the strong indigenous mechanism for DNA double strand break repair; therefore, strengthening of the DNA double strand break repair mechanism with PQQ would have a significant effect on UVC tolerance in E. coli.…”

Section: Resultsmentioning

confidence: 99%

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Involvement of a Protein Kinase Activity Inducer in DNA Double Strand Break Repair and Radioresistance of Deinococcus radiodurans

2008

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Transgenic bacteria producing pyrroloquinoline quinone, a known cofactor for dehydrogenases and an inducer of a periplasmic protein kinase activity, show resistance to both oxidative stress and protection from nonoxidative effects of radiation and DNA-damaging agents. Deinococcus radiodurans R1 encodes an active pyrroloquinoline quinone synthase, and constitutive synthesis of pyrroloquinoline quinone occurred in wildtype bacteria. Disruption of a genomic copy of pqqE resulted in cells that lacked this cofactor. The mutant showed a nearly 3-log decrease in ␥ radiation resistance and a 2-log decrease in mitomycin C tolerance compared to wild-type cells. The mutant cells did not show sensitivity to UVC radiation. Expression of pyrroloquinoline quinone synthase in trans showed that there was functional complementation of ␥ resistance and mitomycin C tolerance in the pqqE mutant. The sensitivity to ␥ radiation was due to impairment or slow kinetics of DNA double strand break repair. Low levels of 32 P incorporation were observed in total soluble proteins of mutant cells compared to the wild type. The results suggest that pyrroloquinoline quinone has a regulatory role as a cofactor for dehydrogenases and an inducer of selected protein kinase activity in radiation resistance and DNA strand break repair in a radioresistant bacterium.Pyrroloquinoline quinone (PQQ) has been shown to be a redox cofactor for periplasmic as well as cytosolic dehydrogenases, contributing to the mineral phosphate solubilization phenotype in bacteria (11). This compound has been reported to act as an antioxidant in vitro (33), in animal systems (13), and in bacterial systems (18) in vivo and as a member of the B group vitamins (16). He and coworkers (13) have shown that the antioxidant nature of PQQ is concentration dependent. Higher concentrations of PQQ induce oxidative stress for mitochondrial activity in rats, which leads to both apoptotic and necrotic cell death. Further studies indicated that the necrotic cell death could be selectively inhibited in the presence of antioxidants, while apoptotic cell death continued by a stillunknown mechanism. Further, a possible role for PQQ as an inducer for proteins kinases involved in distinctly different metabolic and physiological processes has been suggested (20).Deinococcus radiodurans R1, a gram-positive bacterium, exhibits extraordinary tolerance to various abiotic stresses, including radiation, desiccation, and other DNA-damaging factors (3). DNA double strand break repair in D. radiodurans R1 follows biphasic kinetics (8). Phase I is RecA independent and involves an extended synthesis-dependent strand annealing mechanism for reassembly of the fragmented genome (42), while phase II involves RecA-dependent slow crossover events (9). The extreme phenotypes of this bacterium are believed to be due to the presence of an efficient DNA strand break repair mechanism (1, 31) and strong oxidative stress tolerance (27). A comparison of the genome sequence of D. radiodurans R1 (41) with the genome sequenc...

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Section: Resultsmentioning

confidence: 99%

“…Deinococcus cells were treated with different doses of UV and ␥ radiation as described previously (19). In brief, mutant and wild-type D. radiodurans cells and pqqE mutant cells harboring pGropqqE were grown in TGY medium to the late log phase at 32°C.…”

Section: Methodsmentioning

confidence: 99%

Involvement of a Protein Kinase Activity Inducer in DNA Double Strand Break Repair and Radioresistance of Deinococcus radiodurans

2008

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“…The RecBCD heterotrimer acts at the same time as helicase, ATPdependent dsDNA exonuclease, ssDNA exonuclease, and ssDNA endonuclease (143,323). D. radiodurans does not encode RecB and RecC homologs, or SbcB (a 3Ј-5Ј ssDNA nuclease), and the levels of nuclease activity in deinococcal cell extracts are consequently much lower than that in E. coli extracts (288). The overexpression of E. coli RecBC (288) and SbcB (418) in D. radiodurans sensitizes the cells to ionizing radiation and delays or inhibits DSB repair in gamma-irradiated cells, respectively.…”

Section: Recombinational Processes In D Radiodurans Dna Repairmentioning

confidence: 99%

“…D. radiodurans does not encode RecB and RecC homologs, or SbcB (a 3Ј-5Ј ssDNA nuclease), and the levels of nuclease activity in deinococcal cell extracts are consequently much lower than that in E. coli extracts (288). The overexpression of E. coli RecBC (288) and SbcB (418) in D. radiodurans sensitizes the cells to ionizing radiation and delays or inhibits DSB repair in gamma-irradiated cells, respectively. RecBC-expressing D. radiodurans cells suffer from an extensive degradation of DNA ends, while the inhibition of DSB repair in SbcB-expressing cells suggests that 3Ј ssDNA ends are indispensable for the recombinational repair of DSBs (418).…”

Section: Recombinational Processes In D Radiodurans Dna Repairmentioning

confidence: 99%

Oxidative Stress Resistance inDeinococcus radiodurans

Slade

Radman

2011

Microbiol Mol Biol Rev

648

639

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SUMMARY Deinococcus radiodurans is a robust bacterium best known for its capacity to repair massive DNA damage efficiently and accurately. It is extremely resistant to many DNA-damaging agents, including ionizing radiation and UV radiation (100 to 295 nm), desiccation, and mitomycin C, which induce oxidative damage not only to DNA but also to all cellular macromolecules via the production of reactive oxygen species. The extreme resilience of D. radiodurans to oxidative stress is imparted synergistically by an efficient protection of proteins against oxidative stress and an efficient DNA repair mechanism, enhanced by functional redundancies in both systems. D. radiodurans assets for the prevention of and recovery from oxidative stress are extensively reviewed here. Radiation- and desiccation-resistant bacteria such as D. radiodurans have substantially lower protein oxidation levels than do sensitive bacteria but have similar yields of DNA double-strand breaks. These findings challenge the concept of DNA as the primary target of radiation toxicity while advancing protein damage, and the protection of proteins against oxidative damage, as a new paradigm of radiation toxicity and survival. The protection of DNA repair and other proteins against oxidative damage is imparted by enzymatic and nonenzymatic antioxidant defense systems dominated by divalent manganese complexes. Given that oxidative stress caused by the accumulation of reactive oxygen species is associated with aging and cancer, a comprehensive outlook on D. radiodurans strategies of combating oxidative stress may open new avenues for antiaging and anticancer treatments. The study of the antioxidation protection in D. radiodurans is therefore of considerable potential interest for medicine and public health.

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“…The polX deletion mutant was generated using protocols similar to that described earlier (Khairnar et al, 2008). In brief, the sequences 1 kb upstream and 1 kb downstream of polX (DR0467) were PCR amplified from the D. radiodurans genome using NPK1 and NPK2 for downstream and NPK3 and NPK4 for upstream, respectively.…”

Section: Methodsmentioning

confidence: 99%

DNA polymerase X from Deinococcus radiodurans implicated in bacterial tolerance to DNA damage is characterized as a short patch base excision repair polymerase

Khairnar

Misra

2009

Microbiology

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The Deinococcus radiodurans R1 genome encodes an X-family DNA repair polymerase homologous to eukaryotic DNA polymerase b. The recombinant deinococcal polymerase X (PolX) purified from transgenic Escherichia coli showed deoxynucleotidyltransferase activity. Unlike the Klenow fragment of E. coli, this enzyme showed short patch DNA synthesis activity on heteropolymeric DNA substrate. The recombinant enzyme showed 59-deoxyribose phosphate (59-dRP) lyase activity and base excision repair function in vitro, with the help of externally supplied glycosylase and AP endonuclease functions. A polX disruption mutant of D. radiodurans expressing 59-dRP lyase and a truncated polymerase domain was comparatively less sensitive to c-radiation than a polX deletion mutant. Both mutants showed higher sensitivity to hydrogen peroxide. Excision repair mutants of E. coli expressing this polymerase showed functional complementation of UV sensitivity. These results suggest the involvement of deinococcal polymerase X in DNA-damage tolerance of D. radiodurans, possibly by contributing to DNA double-strand break repair and base excision repair.

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RecBC enzyme overproduction affects UV and gamma radiation survival of Deinococcus radiodurans

Cited by 43 publications

References 43 publications

Involvement of a Protein Kinase Activity Inducer in DNA Double Strand Break Repair and Radioresistance of Deinococcus radiodurans

Involvement of a Protein Kinase Activity Inducer in DNA Double Strand Break Repair and Radioresistance of Deinococcus radiodurans

Oxidative Stress Resistance inDeinococcus radiodurans

DNA polymerase X from Deinococcus radiodurans implicated in bacterial tolerance to DNA damage is characterized as a short patch base excision repair polymerase

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