Analysis of interaction between DNA and Deinococcus radiodurans PprA protein by Atomic force microscopy

Murakami, Masahiro; Narumi, Issay; Satoh, Katsuya; Furukawa, Akira; Hayata, Isamu

doi:10.1016/j.bbapap.2005.10.017

Cited by 11 publications

(8 citation statements)

References 23 publications

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“…To our knowledge, the spectacular efficiency of DdrC to circularize linear DNA has never been reported for any other Deinococcus protein, including PprA [39]. However, whereas the PprA protein was able to stimulate DNA ligase activity [13], we did not observe any evidence of the stimulation of DNA ligation by the DdrC protein.…”

Section: Discussioncontrasting

confidence: 71%

In vivo and in vitro characterization of DdrC, a DNA damage response protein in Deinococcus radiodurans bacterium

et al. 2017

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The bacterium Deinococcus radiodurans possesses a set of Deinococcus-specific genes highly induced after DNA damage. Among them, ddrC (dr0003) was recently re-annotated, found to be in the inverse orientation and called A2G07_00380. Here, we report the first in vivo and in vitro characterization of the corrected DdrC protein to better understand its function in irradiated cells. In vivo, the ΔddrC null mutant is sensitive to high doses of UV radiation and the ddrC deletion significantly increases UV-sensitivity of ΔuvrA or ΔuvsE mutant strains. We show that the expression of the DdrC protein is induced after γ-irradiation and is under the control of the regulators, DdrO and IrrE. DdrC is rapidly recruited into the nucleoid of the irradiated cells. In vitro, we show that DdrC is able to bind single- and double-stranded DNA with a preference for the single-stranded DNA but without sequence or shape specificity and protects DNA from various nuclease attacks. DdrC also condenses DNA and promotes circularization of linear DNA. Finally, we show that the purified protein exhibits a DNA strand annealing activity. Altogether, our results suggest that DdrC is a new DNA binding protein with pleiotropic activities. It might maintain the damaged DNA fragments end to end, thus limiting their dispersion and extensive degradation after exposure to ionizing radiation. DdrC might also be an accessory protein that participates in a single strand annealing pathway whose importance in DNA repair becomes apparent when DNA is heavily damaged.

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Section: Discussioncontrasting

confidence: 71%

In vivo and in vitro characterization of DdrC, a DNA damage response protein in Deinococcus radiodurans bacterium

et al. 2017

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“…We first focused on the characteristics of PprA self-association because previous gel filtration results suggested that PprA forms 12-mers, whereas the results with atomic force microscopy indicated that PprA monomers or dimers could bind to DNA. 28 These inconsistencies might be due to differences in the experimental conditions, such as the protein concentration, because the self-association of PprA without DNA significantly depends on the PprA and salt concentrations, as shown in Figures 1 and 2. In addition, it is considered that the large change in molecular weight as PprA concentration increases is important for its function as a pleiotropic protein, because PprA expression is induced following exposure to ionizing radiation.…”

Section: Discussionmentioning

confidence: 99%

“…PprA is a 32-kDa protein that does not show any significant amino acid similarity to other known proteins except PprAs from other members of the genus Deinococcus. 14,[19][20][21][22] The reported molecular characteristics of PprA are as follows: PprA is induced when the bacterium is exposed to ionizing radiation 15,[23][24][25][26][27] ; PprA can bind to DNA 14,28 ; PprA can promote the ligation activities of NADdependent E. coli DNA ligase and ATP-dependent T4 DNA ligase 14 ; and PprA can inhibit DNA degradation by E. coli exonuclease III. 14 These characteristics suggest the possibility of a nonhomologous end joining repair pathway in D. radiodurans.…”

Section: Introductionmentioning

confidence: 99%

“…14,[19][20][21][22] The reported molecular characteristics of PprA are as follows: PprA is induced when the bacterium is exposed to ionizing radiation 15,[23][24][25][26][27] ; PprA can bind to DNA 14,28 31,32 Thus, PprA appears to be a potential pleiotropic protein that functions in the repair of DSBs and other radiation-induced damage. However, the molecular mechanism underlying DNA repair involving PprA remains unclear.…”

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confidence: 99%

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Interaction of double‐stranded DNA with polymerized PprA protein from Deinococcus radiodurans

et al. 2014

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Pleiotropic protein promoting DNA repair A (PprA) is a key protein that facilitates the extreme radioresistance of Deinococcus radiodurans. To clarify the role of PprA in the radioresistance mechanism, the interaction between recombinant PprA expressed in Escherichia coli with several double-stranded DNAs (i.e., super coiled, linear, or nicked circular dsDNA) was investigated. In a gel-shift assay, the band shift of supercoiled pUC19 DNA caused by the binding of PprA showed a bimodal distribution, which was promoted by the addition of 1 mM Mg, Ca, or Sr ions. The dissociation constant of the PprA-supercoiled pUC19 DNA complex, calculated from the relative portions of shifted bands, was 0.6 lM with Hill coefficient of 3.3 in the presence of 1 mM Mg acetate. This indicates that at least 281 PprA molecules are required to saturate a supercoiled pUC19 DNA, which is consistent with the number (280) of bound PprA molecules estimated by the UV absorption of the PprA-pUC19 complex purified by gel filtration. This saturation also suggests linear polymerization of PprA along the dsDNA. On the other hand, the bands of linear dsDNA and nicked circular dsDNA that eventually formed PprA complexes did not saturate, but created larger molecular complexes when the PprA concentration was >1.3 lM. This result implies that DNAbound PprA aids association of the termini of damaged DNAs, which is regulated by the concentration of PprA. These findings are important for the understanding of the mechanism underlying effective DNA repair involving PprA.

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“…In vitro, PprA stimulates DNA end-joining reactions catalyzed by ATP-and NAD-dependent DNA ligases while inhibiting E. coli exonuclease III activity and may thus protect DNA ends from extensive degradation (456). It binds DNA ends with a greater affinity than internal DNA regions and may even promote DNA looping (451). Its ability to bind dsDNA carrying strand breaks was harnessed to visualize radiation-induced DNA strand breaks in mammalian cell cultures by immunofluorescence, where permeabilized irradiated cells were treated with PprA and fluorescently labeled with an anti-PprA antibody (538).…”

Section: Recombinational Processes In D Radiodurans Dna Repairmentioning

confidence: 99%

Oxidative Stress Resistance inDeinococcus radiodurans

Slade

Radman

2011

Microbiol Mol Biol Rev

650

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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Analysis of interaction between DNA and Deinococcus radiodurans PprA protein by Atomic force microscopy

Cited by 11 publications

References 23 publications

In vivo and in vitro characterization of DdrC, a DNA damage response protein in Deinococcus radiodurans bacterium

In vivo and in vitro characterization of DdrC, a DNA damage response protein in Deinococcus radiodurans bacterium

Interaction of double‐stranded DNA with polymerized PprA protein from Deinococcus radiodurans

Oxidative Stress Resistance inDeinococcus radiodurans

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