Structural and functional insights into DNA-end processing by the archaeal HerA helicase–NurA nuclease complex

Blackwood, John K.; Rzechorzek, Neil J.; Abrams, Andrew S.; Maman, Joseph D.; Pellegrini, Luca; Robinson, Nicholas P.

doi:10.1093/nar/gkr1157

Cited by 41 publications

(127 citation statements)

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“…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: 89%

“…It has been shown that the channel formed by two protomers is too narrow to accommodate B-form dsDNA, but it might be suited to adjust one or two strands of an unwound duplex (14,43). NurA secondary structure analysis suggests a low level of sequence similarity between paralogous and orthologous groups (18).…”

Section: Resultsmentioning

confidence: 99%

“…The sequences of NurA and HerA proteins from D. radiodurans, Sulfolobus solfataricus, Pyrococcus furiosus, Thermotoga maritima, and other species were obtained from the NCBI website. Motif and active site predictions of HerA were based on an earlier studies of HerA from Methanobacter thermoautotrophicus (17), Sulfolobus acidocaldarius (16), P. furiosus (13), and S. solfataricus (43) and the TrwB protein (EcoTrwB) and FtsK protein (EcoFtsK) from E. coli (44,45). The DrHerA three-dimensional structure was modeled based on the S. solfataricus HerA (SsoHerA) structure (PDB 4D2I) by using the Phyre2 online program (http://www.sbg.bio.ic.ac.uk /phyre2) (46).…”

Section: Methodsmentioning

confidence: 99%

“…The DrHerA three-dimensional structure was modeled based on the S. solfataricus HerA (SsoHerA) structure (PDB 4D2I) by using the Phyre2 online program (http://www.sbg.bio.ic.ac.uk /phyre2) (46). Motif and active site predictions of NurA were based on earlier studies of P. furiosus NurA (PfuNurA; PDB 3TAI) (13,14) and S. solfataricus NurA (SsoNurA; PDB 2YGK) (43). Multiple alignments of HerA and NurA were performed by using the Cobalt constraint-based multiple protein alignment tool on the NCBI website (http://www.ncbi .nlm.nih.gov/tools/cobalt), followed by manual corrections.…”

Section: Methodsmentioning

confidence: 99%

“…Physical interactions between HerA (wild type and C-terminal domain) and NurA were investigated by using analytical gel filtration as previously described with some modification (43). HerA alone (6 mol; protomer), NurA alone (3 mol; protomer), HerA-C alone (6 mol; protomer), or HerA (6 mol; protomer) and NurA (3 mol; protomer), HerA-C (6 mol; protomer), and NurA (3 mol; protomer) were mixed and preincubated at 30°C for 20 min in a total 300 l of gel filtration buffer (20 mM Tris [pH 8.0], 150 mM NaCl, 5% glycerol, 1 mM DTT, 1 mM EDTA).…”

Section: Methodsmentioning

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: Resultsmentioning

confidence: 89%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

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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The Family Sulfolobaceae

Albers¹,

Siebers²

2014

The Prokaryotes

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The Sulfolobus solfataricus RecQ-like DNA helicase Hel112 inhibits the NurA/HerA complex exonuclease activity

et al. 2018

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ATPase/Helicases and nucleases play important roles in DNA end-resection, a critical step during homologous recombination repair in all organisms. In hyperthermophilic archaea the exo-endonuclease NurA and the ATPase HerA cooperate with the highly conserved Mre11-Rad50 complex in 3' single-stranded DNA (ssDNA) end processing to coordinate repair of double-stranded DNA breaks. Little is known, however, about the assembly mechanism and activation of the HerA-NurA complex. In this study we demonstrate that the NurA exonuclease activity is inhibited by the Sulfolobus solfataricus RecQ-like Hel112 helicase. Inhibition occurs both in the presence and in the absence of HerA, but is much stronger when NurA is in complex with HerA. In contrast, the endonuclease activity of NurA is not affected by the presence of Hel112. Taken together these results suggest that the functional interaction between NurA/HerA and Hel112 is important for DNA end-resection in archaeal homologous recombination.

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Structural and functional insights into DNA-end processing by the archaeal HerA helicase–NurA nuclease complex

Cited by 41 publications

References 50 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

The Family Sulfolobaceae

The Sulfolobus solfataricus RecQ-like DNA helicase Hel112 inhibits the NurA/HerA complex exonuclease activity

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