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Crystal structure of the NurA-dAMP-Mn2+ complex.

ABSTRACT: Generation of the 3' overhang is a critical event during homologous recombination (HR) repair of DNA double strand breaks. A 5'-3' nuclease, NurA, plays an important role in generating 3' single-stranded DNA during archaeal HR, together with Mre11-Rad50 and HerA. We have determined the crystal structures of apo- and dAMP-Mn(2)(+)-bound NurA from Pyrococcus furiousus (Pf?NurA) to provide the basis for its cleavage mechanism. Pf?NurA forms a pyramid-shaped dimer containing a large central channel on one side, which becomes narrower towards the peak of the pyramid. The structure contains a PIWI domain with high similarity to argonaute, endoV nuclease and RNase H. The two active sites, each of which contains Mn(2)(+) ion(s) and dAMP, are at the corners of the elliptical channel near the flat face of the dimer. The 3' OH group of the ribose ring is directed toward the channel entrance, explaining the 5'-3' nuclease activity of Pf?NurA. We provide a DNA binding and cleavage model for Pf?NurA.

SUBMITTER: Chae J

PROVIDER: S-EPMC3300031 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Crystal structure of the NurA-dAMP-Mn2+ complex.

Chae Jina J Kim Young Chang YC Cho Yunje Y

Nucleic acids research 20111107 5

Generation of the 3' overhang is a critical event during homologous recombination (HR) repair of DNA double strand breaks. A 5'-3' nuclease, NurA, plays an important role in generating 3' single-stranded DNA during archaeal HR, together with Mre11-Rad50 and HerA. We have determined the crystal structures of apo- and dAMP-Mn(2)(+)-bound NurA from Pyrococcus furiousus (Pf NurA) to provide the basis for its cleavage mechanism. Pf NurA forms a pyramid-shaped dimer containing a large central channel ...[more]

PMID: 22064858

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Project description:Crystals of Zn2+/Mn2+ yeast enolase with the inhibitor PhAH (phosphonoacetohydroxamate) were grown under conditions with a slight preference for binding of Zn2+ at the higher affinity site, site I. The structure of the Zn2+/Mn2+-PhAH complex was solved at a resolution of 1.54 A, and the two catalytic metal binding sites, I and II, show only subtle displacement compared to that of the corresponding complex with the native Mg2+ ions. Low-temperature echo-detected high-field (W-band, 95 GHz) EPR (electron paramagnetic resonance) and 1H ENDOR (electron-nuclear double resonance) were carried out on a single crystal, and rotation patterns were acquired in two perpendicular planes. Analysis of the rotation patterns resolved a total of six Mn2+ sites, four symmetry-related sites of one type and two out of the four of the other type. The observation of two chemically inequivalent Mn2+ sites shows that Mn2+ ions populate both sites I and II and the zero-field splitting (ZFS) tensors of the Mn2+ in the two sites were determined. The Mn2+ site with the larger D value was assigned to site I based on the 1H ENDOR spectra, which identified the relevant water ligands. This assignment is consistent with the seemingly larger deviation of site I from octahedral symmetry, compared to that of site II. The ENDOR results gave the coordinates of the protons of two water ligands, and adding them to the crystal structure revealed their involvement in a network of H bonds stabilizing the binding of the metal ions and PhAH. Although specific hyperfine interactions with the inhibitor were not determined, the spectroscopic properties of the Mn2+ in the two sites were consistent with the crystal structure. Density functional theory (DFT) calculations carried out on a cluster representing the catalytic site, with Mn2+ in site I and Zn2+ in site II, and vice versa, gave overestimated D values on the order of the experimental ones, although the larger D value was found for Mn2+ in site II rather than in site I. This discrepancy was attributed to the high sensitivity of the ZFS parameters to the Mn-O bond lengths and orientations, such that small, but significant, differences between the optimized and crystal structures alter the ZFS considerably, well above the difference between the two sites.

Project description:UnlabelledIn archaea, the NurA nuclease and HerA ATPase/helicase, together with the Mre11-Rad50 complex, function in 3' single-stranded 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.

Dataset Information

Crystal structure of the NurA-dAMP-Mn2+ complex.

Publications

Crystal structure of the NurA-dAMP-Mn2+ complex.

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