Structure of the hexameric HerA ATPase reveals a mechanism of translocation-coupled DNA-end processing in archaea

Rzechorzek, Neil J.; Blackwood, John K.; Bray, Sian; Maman, Joseph D.; Pellegrini, Luca; Robinson, Nicholas P.

doi:10.1038/ncomms6506

Cited by 44 publications

(68 citation statements)

References 58 publications

(129 reference statements)

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“…DrHerA alone (67.4 kDa per protomer) eluted at around 10 ml on Superdex 200 as hexamers (Fig. 3C), consistent with earlier reports for M. thermoautotrophicus (17) and S. solfataricus (43) and the SsoHerA structure (53,54). DrNurA (38.3 kDa per protomer) alone eluted around 14 ml on Superdex 200, indicating a monomer-dimer equilibrium (Fig.…”

Section: Resultssupporting

confidence: 78%

“…A previous S. solfataricus study showed that HerA and NurA interacted with a 6:2 stoichiometry (43), consistent with the crystal structure of the NurA-HerA complex (53,54). We purified DrHerA and DrNurA separately for analysis by gel filtration chromatography (see Fig.…”

Section: Resultsmentioning

confidence: 71%

“…radiodurans NurA and HerA interact in vivo and in vitro. Although no direct interaction between HerA and NurA was reported for S. acidocaldarius (21), interactions have been confirmed for S. solfataricus (43,53,54) and P. furiosus (13). Based on the conserved operon clustering of NurA and HerA in D. radiodurans, we hypothesized that DrNurA and DrHerA might physically or functionally interact with each other.…”

Section: Resultsmentioning

confidence: 99%

“…Amino acid sequence alignment using far evolutionary distance-originated proteins was not an accurate prediction method, so we built a homology DrHerA model based on the SsoHerA structure (PDB 4D2I) (Phyre2 online program [46]) (53). By comparing the two HerA promoter structures, we found that a major difference between DrHerA and SsoHerA might be a longer helix bundle area and a less-structured flexible loop area in DrHerA (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…2A). To analyze the biochemical function of DrHerA, we identified several key conserved residues by using published structural and biochemical information (53).…”

Section: Resultsmentioning

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: Resultssupporting

confidence: 78%

Section: Resultsmentioning

confidence: 71%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

“…2A). To analyze the biochemical function of DrHerA, we identified several key conserved residues by using published structural and biochemical information (53).…”

Section: Resultsmentioning

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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Nucleotide‐dependent conformational changes of the AAA+ ATPase p97 revisited

et al. 2016

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Edited by Peter BrzezinskiThe ubiquitous AAA-ATPase p97 segregates ubiquitylated proteins from their molecular environment. Previous studies of the nucleotide-dependent conformational changes of p97 were inconclusive. Here, we determined its structure in the presence of ADP, AMP-PNP, or ATP-cS at 6.1-7.4 A resolution using single particle cryo-electron microscopy. Both AAA domains, D1 and D2, assemble into essentially six-fold symmetrical rings. The pore of the D1-ring remains essentially closed under all nucleotide conditions, whereas the D2-ring shows an iris-like opening for ADP. The largest conformational changes of p97 are 'swinging motions' of the N-terminal domains, which may enable segregation of ubiquitylated substrates from their environment.

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