Function and biochemical characterization of RecJ in Deinococcus radiodurans

Jiao, Jing; Wang, Liangyan; Xia, Wenrong; Li, Mingfeng; Sun, Hongmin; Xu, Guangzhi; Teng, Bing; Hua, Yuejin

doi:10.1016/j.dnarep.2011.11.008

Cited by 22 publications

(32 citation statements)

References 61 publications

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“…Knockout of nurA, herA, or the nurA-herA operon was carried out using a deletion replacement method described previously (35,50). The upstream and downstream target genes with BamHI and HindIII digestion sites, respectively, were amplified by PCR.…”

Section: Methodsmentioning

confidence: 99%

“…Growth curve and temperature sensitive assays were performed as previously described (35). Briefly, after the cell density of the culture (OD 600 ) reached 1.0, 1-ml aliquots were resuspended in 100 ml new fresh TGY medium and incubated at 30°C or 37°C.…”

Section: Methodsmentioning

confidence: 99%

“…An ATPase assay was performed as previously described (45), which was measured by spectrophotometric detection of inorganic phosphate (P i ) with malachite green reagent 35,40,45,50,55, and 60°C were used.) Malachite green reagent (200 ml in 700 ml water) was added, and P i release was monitored at 630 nm.…”

Section: Methodsmentioning

confidence: 99%

“…In Deinococcus radiodurans, RecJ has 5=-to-3= ssDNA exonuclease activity (35). However, knocking out recJ only modestly decreases radioresistance, indicating that another nuclease might be involved in 3=-end processing in HR (35). Although RecQ, HelD, and RecD2 all possess DNA helicase activity (36)(37)(38), none of them makes a remarkable contribution to robust DNA repair in D. radiodurans (37,39,40).…”

mentioning

confidence: 99%

“…These bacteria use the RecFOR pathway as the major recombinational DNA repair pathway and naturally lack RecBCD/AddAB systems (31,34). In Deinococcus radiodurans, RecJ has 5=-to-3= ssDNA exonuclease activity (35). However, knocking out recJ only modestly decreases radioresistance, indicating that another nuclease might be involved in 3=-end processing in HR (35).…”

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confidence: 99%

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Biochemical and Functional Characterization of the NurA-HerA Complex from Deinococcus radiodurans

Cheng

Chen

et al. 2015

J Bacteriol

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In archaea, the NurA nuclease and HerA ATPase/helicase, together with the Mre11-Rad50 complex, function in 3= singlestranded DNA (ssDNA) end processing during homologous recombination (HR). However, bacterial homologs of NurA and HerA have not been characterized. From Deinococcus radiodurans, we identified the manganese-dependent 5=-to-3= ssDNA/double-stranded DNA (dsDNA) exonuclease/endonuclease NurA (DrNurA) and the ATPase HerA (DrHerA). These two proteins stimulated each other's activity through direct protein-protein interactions. The N-terminal HAS domain of DrHerA was the key domain for this interaction. Several critical residues of DrNurA and DrHerA were verified by site-directed mutational analysis. Temperature-dependent activity assays confirmed that the two proteins had mesophilic features, with optimum activity temperatures 10°C to 15°C higher than their optimum growth temperatures. Knocking out either nurA or herA affected cell proliferation by shortening the growth phase, especially for growth at a high temperature (37°C). In addition, both mutant strains displayed almost 10-fold-reduced intermolecular recombination efficiency, indicating that DrNurA and DrHerA might be involved in homologous recombination in vivo. However, single-and double-gene deletions did not show significantly decreased radioresistance. Our results confirmed that the biochemical activities of bacterial NurA and HerA proteins were conserved with archaea. Our phenotypical results suggested that these proteins might have different functions in bacteria. IMPORTANCEDeinococcus radiodurans NurA (DrNurA) was identified as a manganese-dependent 5=-to-3= ssDNA/dsDNA exonuclease/endonuclease, and Deinococcus radiodurans HerA (DrHerA) was identified as an ATPase. Physical interactions between DrNurA and DrHerA explained mutual stimulation of their activities. The N-terminal HAS domain on DrHerA was identified as the interaction domain. Several essential functional sites on DrNurA and DrHerA were characterized. Both DrHerA and DrNurA showed mesophilic biochemical features, with their optimum activity temperatures 10°C to 15°C higher than their optimum growth temperatures in vitro. Knockout of nurA or herA led to abnormal cell proliferation and reduced intermolecular recombination efficiency but no obvious effect on radioresistence.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Biochemical and Functional Characterization of the NurA-HerA Complex from Deinococcus radiodurans

Cheng

Chen

et al. 2015

J Bacteriol

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RecJ from Bacillus halodurans possesses endonuclease activity at moderate temperature

Wen

Zheng

et al. 2020

FEBS Letters

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RecJ homologs, which occur in virtually all prokaryotes and eukaryotes, play key roles in DNA damage repair and recombination. Current evidence shows that RecJ family proteins exhibit exonuclease activity, degrading single‐stranded nucleic acids. Here, we report a novel RecJ isolated from Bacillus halodurans, which utilizes double‐stranded DNA as a substrate and functions as an endonuclease. Bacillus halodurans RecJ (BhRecJ) cleaves supercoiled plasmids into open circular and linear forms. Besides the typical domains of DHH, DHHA1, and oligonucleotide‐binding‐fold, BhRecJ possesses a C‐terminal domain with unknown function, which might form the core of the endonuclease activity. Using mutational analysis, we mapped several essential residues for BhRecJ endonuclease activity. Our findings suggest that BhRecJ may be involved in biological processes not typically associated with RecJ proteins.

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Deinococcus radiodurans YgjD and YeaZ are involved in the repair of DNA cross-links

et al. 2012

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Orthologs of Escherichia coli ygjD and yeaZ genes are highly conserved in various organisms. The genome of the radioresistant bacterium Deinococcus radiodurans possesses single orthologs of ygjD (DR_0382) and yeaZ (DR_0756). Complete loss of either one or both genes did not result in any significant changes in cell growth efficiency, indicating that both genes are not essential for cell viability in D. radiodurans, unlike the case with other species such as E. coli, Bacillus subtilis and Saccharomyces cerevisiae. Survival rates following DNA damage induced by hydrogen peroxide (H(2)O(2)), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ultra violet (UV) radiation, γ-rays, cisplatin and mitomycin C (MMC) were compared among the wild-type strain and D. radiodurans ygjD/yeaZ null mutants. Cell viability of the null mutants did not decrease following exposure to H(2)O(2) or MNNG. In addition, the reduction in cell viability following exposure to γ-rays, UV radiation or cisplatin was marginal in the null mutants compared to the wild-type strain. Interestingly, the null mutants exhibited high sensitivity to MMC, which mainly causes interstrand DNA cross-links. The sensitivity of the null mutants to MMC was restored to that of the wild type by transformation with plasmids expressing these genes. These results suggest that D. radiodurans ygjD and yeaZ genes are involved in DNA repair and play a role in the repair of DNA cross-links.

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Function and biochemical characterization of RecJ in Deinococcus radiodurans

Cited by 22 publications

References 61 publications

Biochemical and Functional Characterization of the NurA-HerA Complex from Deinococcus radiodurans

Biochemical and Functional Characterization of the NurA-HerA Complex from Deinococcus radiodurans

RecJ from Bacillus halodurans possesses endonuclease activity at moderate temperature

Deinococcus radiodurans YgjD and YeaZ are involved in the repair of DNA cross-links

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