Biochemical characterization of RecA variants that contribute to extreme resistance to ionizing radiation

Piechura, Joseph Robert; Tseng, Tzu‐Ling; Hsu, Hsin-Fang; Byrne, Rose T.; Windgassen, Tricia A.; Chitteni-Pattu, Sindhu; Battista, John R.; Li, Hung‐Wen; Cox, Michael M.

doi:10.1016/j.dnarep.2014.12.001

Cited by 24 publications

(31 citation statements)

References 89 publications

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“…Although the filament nucleation process characterized in the studies by Piechura et al, is a much more composite process than a simple on-rate for DNA binding, it is still intriguing to note that a faster on-rate in a binding equilibrium process, in the absence of any offrate changes, would produce tighter binding (Kd = off-rate/on-rate). Likewise, the shorter filaments formed by the radiation-resistant EcRecA mutants [32], like the shorter filaments formed by DrRecA itself [6], mirror the intrinsically shorter free-filament formation by DrRecA suggested in the present study.…”

Section: Discussionsupporting

confidence: 63%

“…Although no direct binding experiments were performed, the radiation-resistance induced EcRecA D276 mutants found by Hsu et al, like DrRecA itself [6], exhibited both (1) faster filament nucleation on DNA and (2) shorter filament formation [32]. Although the filament nucleation process characterized in the studies by Piechura et al, is a much more composite process than a simple on-rate for DNA binding, it is still intriguing to note that a faster on-rate in a binding equilibrium process, in the absence of any offrate changes, would produce tighter binding (Kd = off-rate/on-rate).…”

Section: Discussionmentioning

confidence: 95%

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Enhanced DNA binding affinity of RecA protein from Deinococcus radiodurans

Warfel

LiCata

2015

DNA Repair

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Deinococcus radiodurans (Dr) has a significantly more robust DNA repair response than Escherichia coli (Ec), which helps it survive extremely high doses of ionizing radiation and prolonged periods of desiccation. DrRecA protein plays an essential part in this DNA repair capability. In this study we directly compare the binding of DrRecA and EcRecA to the same set of short, defined single (ss) and double stranded (ds) DNA oligomers. In the absence of cofactors (ATPγS or ADP), DrRecA binds to dsDNA oligomers more than 20 fold tighter than EcRecA, and binds ssDNA up to 9 fold tighter. Binding to dsDNA oligomers in the absence of cofactor presumably predominantly monitors DNA end binding, and thus suggests a significantly higher affinity of DrRecA for ds breaks. Upon addition of ATPγS, this species-specific affinity difference is nearly abolished, as ATPγS significantly decreases the affinity of DrRecA for DNA. Other findings include that: (1) both proteins exhibit a dependence of binding affinity on the length of the ssDNA oligomer, but not the dsDNA oligomer; (2) the salt dependence of binding is modest for both species of RecA, and (3) in the absence of DNA, DrRecA produces significantly shorter and/or fewer free-filaments in solution than does EcRecA. The results suggest intrinsic biothermodynamic properties of DrRecA contribute directly to the more robust DNA repair capabilities of D. radiodurans.

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Section: Discussionsupporting

confidence: 63%

Section: Discussionmentioning

confidence: 95%

Enhanced DNA binding affinity of RecA protein from Deinococcus radiodurans

Warfel

LiCata

2015

DNA Repair

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“…Among many accessory proteins regulating homologous recombination, the Swi5-Sfr1 complex stimulates various stages of the process, including filament assembly and strand exchange (25,26,(28)(29)(30)(31)(32)(33)(34)(35). Here, we took advantage of previously developed singlemolecule TPM experiments (36)(37)(38) to monitor Rad51 nucleoprotein filament assembly in real time (Fig. 1A).…”

Section: Resultsmentioning

confidence: 99%

Swi5–Sfr1 stimulates Rad51 recombinase filament assembly by modulating Rad51 dissociation

Yeh

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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Eukaryotic Rad51 protein is essential for homologous-recombination repair of DNA double-strand breaks. Rad51 recombinases first assemble onto single-stranded DNA to form a nucleoprotein filament, required for function in homology pairing and strand exchange. This filament assembly is the first regulation step in homologous recombination. Rad51 nucleation is kinetically slow, and several accessory factors have been identified to regulate this step. Swi5–Sfr1 (S5S1) stimulates Rad51-mediated homologous recombination by stabilizing Rad51 nucleoprotein filaments, but the mechanism of stabilization is unclear. We used single-molecule tethered particle motion experiments to show that mouse S5S1 (mS5S1) efficiently stimulates mouse RAD51 (mRAD51) nucleus formation and inhibits mRAD51 dissociation from filaments. We also used single-molecule fluorescence resonance energy transfer experiments to show that mS5S1 promotes stable nucleus formation by specifically preventing mRAD51 dissociation. This leads to a reduction of nucleation size from three mRAD51 to two mRAD51 molecules in the presence of mS5S1. Compared with mRAD51, fission yeast Rad51 (SpRad51) exhibits fast nucleation but quickly dissociates from the filament. SpS5S1 specifically reduces SpRad51 disassembly to maintain a stable filament. These results clearly demonstrate the conserved function of S5S1 by primarily stabilizing Rad51 on DNA, allowing both the formation of the stable nucleus and the maintenance of filament length.

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“…These findings suggest that RecA-mediated DNA repair mechanisms may differ from one Deinococcus species to another. Piechura et al demonstrated that RecA mutations affecting the Asp residue at positions 276 (D276), D276A and D276N increase radiation resistance of E. coli mutant strains up to 3 kGy (Piechura et al 2015). Further studies should be performed to determine if mutations at equivalent sites on RAD51, the eukaryotic homolog of RecA, have a similar effect on radiation resistance.…”

Section: Proteins Involved In Genome Preservation and Dna Repair Mechmentioning

confidence: 98%

Recent progress in understanding the molecular mechanisms of radioresistance in Deinococcus bacteria

2015

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The deleterious effects of ionizing radiation are a major concern of the modern world. In the last decades, outstanding interest has been given to developing new therapeutic tools designed for protection against the toxic effects of ionizing radiation. Deinococcus spp. are among the most radioresistant organisms on Earth, being able to survive extreme doses of radiation, 1000-fold higher than most vertebrates. The molecular mechanisms underlying DNA repair and biomolecular protection, which are responsible for the remarkable radioresistance of Deinococcus bacteria, have been a debatable subject for the last 60 years. This paper is focused on the most recent findings regarding the molecular background of radioresistance and on Deinococcus bacteria response to oxidative stress. Novel proteins and genes involved in the highly regulated DNA repair processes, and enzymatic and non- enzymatic antioxidant systems are presented. In addition, a recently proposed mechanism that may contribute to oxidative damage protection in Deinococcus bacteria is discussed. A better understanding of these molecular mechanisms may draw future perspectives for counteracting radiation-related toxicity.

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Biochemical characterization of RecA variants that contribute to extreme resistance to ionizing radiation

Cited by 24 publications

References 89 publications

Enhanced DNA binding affinity of RecA protein from Deinococcus radiodurans

Enhanced DNA binding affinity of RecA protein from Deinococcus radiodurans

Swi5–Sfr1 stimulates Rad51 recombinase filament assembly by modulating Rad51 dissociation

Recent progress in understanding the molecular mechanisms of radioresistance in Deinococcus bacteria

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