Biochemical characterization of INTS3 and C9ORF80, two subunits of hNABP1/2 heterotrimeric complex in nucleic acid binding.

Human nucleic acid-binding protein 1 and 2 (hNABP1 and hNABP2, also known as hSSB2 and hSSB1 respectively) form two separate and independent complexes with two identical proteins, integrator complex subunit 3 (INTS3) and C9ORF80. We and other groups have demonstrated that hNABP1 and 2 are single-stranded (ss) DNA- and RNA-binding proteins, and function in DNA repair; however, the function of INTS3 and C9OFR80 remains elusive. In the present study, we purified recombinant proteins INTS3 and C9ORF80 to near homogeneity. Both proteins exist as a monomer in solution; however, C9ORF80 exhibits anomalous behavior on SDS-PAGE and gel filtration because of 48% random coil present in the protein. Using electrophoretic mobility shift assay (EMSA), INTS3 displays higher affinity toward ssRNA than ssDNA, and C9ORF80 binds ssDNA but not ssRNA. Neither of them binds dsDNA, dsRNA, or RNA : DNA hybrid. INTS3 requires minimum of 30 nucleotides, whereas C9OFR80 requires 20 nucleotides for its binding, which increased with the increasing length of ssDNA. Interestingly, our GST pulldown results suggest that the N-terminus of INTS3 is involved in protein-protein interaction, while EMSA implies that the C-terminus is required for nucleic acid binding. Furthermore, we purified the INTS3-hNABP1/2-C9ORF80 heterotrimeric complex. It exhibits weaker binding compared with the individual hNABP1/2; interestingly, the hNABP1 complex prefers ssDNA, whereas hNABP2 complex prefers ssRNA. Using reconstituted heterotrimeric complex from individual proteins, EMSA demonstrates that INTS3, but not C9ORF80, affects the nucleic acid-binding ability of hNABP1 and hNABP2, indicating that INTS3 might regulate hNABP1/2's biological function, while the role of C9ORF80 remains unknown.

C-termini are essential and distinct for nucleic acid binding of human NABP1 and NABP2.

BACKGROUND: Human Nucleic Acid Binding Protein 1 and 2 (hNABP1 and 2; also known as hSSB2 and 1, respectively) are two newly identified single-stranded (ss) DNA binding proteins (SSB). Both NABP1 and NABP2 have a conserved oligonucleotide/oligosaccharide-binding (OB)-fold domain and a divergent carboxy-terminal domain, the functional importance of which is unknown. METHODS: Recombinant hNABP1/2 proteins were purified using affinity and size exclusion chromatography and their identities confirmed by mass spectrometry. Oligomerization state was checked by sucrose gradient centrifugation. Secondary structure was determined by circular dichroism spectroscopy. Nucleic acid binding ability was examined by EMSA and ITC. RESULTS: Both hNABP1 and hNABP2 exist as monomers in solution; however, hNABP2 exhibits anomalous behavior. CD spectroscopy revealed that the C-terminus of hNABP2 is highly disordered. Deletion of the C-terminal tail diminishes the DNA binding ability and protein stability of hNABP2. Although both hNABP1 and hNABP2 prefer to bind ssDNA than double-stranded (ds) DNA, hNABP1 has a higher affinity for ssDNA than hNABP2. Unlike hNABP2, hNABP1 protein binds and multimerizes on ssDNA with the C-terminal tail responsible for its multimerization. Both hNABP1 and hNABP2 are able to bind single-stranded RNA, with hNABP2 having a higher affinity than hNABP1. CONCLUSIONS: Biochemical evidence suggests that the C-terminal region of NABP1 and NABP2 is essential for their functionality and may lead to different roles in DNA and RNA metabolism. GENERAL SIGNIFICANCE: This is the first report demonstrating the regulation and functional properties of the C-terminal domain of hNABP1/2, which might be a general characteristic of OB-fold proteins.

RAD51 protein ATP cap regulates nucleoprotein filament stability.

RAD51 mediates homologous recombination by forming an active DNA nucleoprotein filament (NPF). A conserved aspartate that forms a salt bridge with the ATP γ-phosphate is found at the nucleotide-binding interface between RAD51 subunits of the NPF known as the ATP cap. The salt bridge accounts for the nonphysiological cation(s) required to fully activate the RAD51 NPF. In contrast, RecA homologs and most RAD51 paralogs contain a conserved lysine at the analogous structural position. We demonstrate that substitution of human RAD51(Asp-316) with lysine (HsRAD51(D316K)) decreases NPF turnover and facilitates considerably improved recombinase functions. Structural analysis shows that archaebacterial Methanococcus voltae RadA(D302K) (MvRAD51(D302K)) and HsRAD51(D316K) form extended active NPFs without salt. These studies suggest that the HsRAD51(Asp-316) salt bridge may function as a conformational sensor that enhances turnover at the expense of recombinase activity.

Crystal structure of an archaeal Rad51 homologue in complex with a metatungstate inhibitor.

Archaeal RadAs are close homologues of eukaryal Rad51s ( approximately 40% sequence identities). These recombinases promote a hallmark strand exchange process between homologous single-stranded and double-stranded DNA substrates. This DNA-repairing function also plays a key role in cancer cells' resistance to chemo- and radiotherapy. Inhibition of the strand exchange process may render cancer cells more susceptible to therapeutic treatment. We found that metatungstate is a potent inhibitor of RadA from Methanococcus voltae. The tungsten cluster binds RadA in the axial DNA-binding groove. This polyanionic species appears to inhibit RadA by locking the protein in its inactive conformation.

Conservation of a conformational switch in RadA recombinase from Methanococcus maripaludis.

Archaeal RadAs are close homologues of eukaryal Rad51s ( approximately 40% sequence identity). These recombinases promote ATP hydrolysis and a hallmark strand-exchange reaction between homologous single-stranded and double-stranded DNA substrates. Pairing of the 3'-overhangs located at the damaged DNA with a homologous double-stranded DNA enables the re-synthesis of the damaged region using the homologous DNA as the template. In recent studies, conformational changes in the DNA-interacting regions of Methanococcus voltae RadA have been correlated with the presence of activity-stimulating potassium or calcium ions in the ATPase centre. The series of crystal structures of M. maripaludis RadA presented here further suggest the conservation of an allosteric switch in the ATPase centre which controls the conformational status of DNA-interacting loops. Structural comparison with the distant Escherichia coli RecA homologue supports the notion that the conserved Lys248 and Lys250 residues in RecA play a role similar to that of cations in RadA. The conservation of a cationic bridge between the DNA-interacting L2 region and the terminal phosphate of ATP, together with the apparent stability of the nucleoprotein filament, suggests a gap-displacement model which may explain the advantage of ATP hydrolysis for DNA-strand exchange.

Crystal structure of Bacillus cereus D-alanyl carrier protein ligase (DltA) in complex with ATP.

D-alanylation of lipoteichoic acids modulates the surface charge and ligand binding of the Gram-positive cell wall. Disruption of the bacterial dlt operon involved in teichoic acid alanylation, as well as inhibition of the DltA (D-alanyl carrier protein ligase) protein, has been shown to render the bacterium more susceptible to conventional antibiotics and host defense responses. The DltA catalyzes the adenylation and thiolation reactions of d-alanine. This enzyme belongs to a superfamily of AMP-forming domains such as the ubiquitous acetyl-coenzyme A synthetase. We have determined the 1.9-A-resolution crystal structure of a DltA protein from Bacillus cereus in complex with ATP. This structure sheds light on the geometry of the bound ATP. The invariant catalytic residue Lys492 appears to be mobile, suggesting a molecular mechanism of catalysis for this superfamily of enzymes. Specific roles are also revealed for two other invariant residues: the divalent cation-stabilizing Glu298 and the beta-phosphate-interacting Arg397. Mutant proteins with a glutamine substitution at position 298 or 397 are inactive.

Crystal structure and enantiomer selection by D-alanyl carrier protein ligase DltA from Bacillus cereus.

Ubiquitous D-alanylation of lipoteichoic acids modulates the surface charge and ligand binding of the gram-positive cell wall. Disruption of the bacterial DltABCD gene involved in teichoic acid alanylation, as well as inhibition of the DltA protein, has been shown to increase a gram-positive bacterium's susceptibility to antibiotics. The DltA D-alanyl carrier protein ligase promotes a two-step process starting with adenylation of D-alanine. We have determined the 2.0 A resolution crystal structure of a DltA protein from Bacillus cereus in complex with the D-alanine adenylate intermediate of the first reaction. Despite the low level of sequence similarity, the DltA structure resembles known structures of adenylation domains such as the acetyl-CoA synthetase. The enantiomer selection appears to be enhanced by the medium-sized side chain of Cys-269. The Ala-269 mutant protein shows marked loss of such selection. The network of noncovalent interactions between the D-alanine adenylate and DltA provides structure-based rationale for aiding the design of tight-binding DltA inhibitors for combating infectious gram-positive bacteria such as the notorious methicillin-resistant Staphylococcus aureus.

Binding of a second magnesium is required for ATPase activity of RadA from Methanococcus voltae.

RecA-like strand exchange proteins, which include closely related archaeal Rad51/RadA and eukaryal Rad51 and DMC1, play a key role in DNA repair by forming helical nucleoprotein filaments which promote a hallmark strand exchange reaction between homologous DNA substrates. Our recent crystallographic studies on a RadA recombinase from Methanococcus voltae (MvRadA) have unexpectedly revealed a secondary magnesium at the subunit interface approximately 11 A from the primary one coordinated by ATP and the canonical P-loop. The DNA-dependent ATPase activity of MvRadA appears to be dependent on the concentration of free Mg2+, while the strand exchange activity does not. We also made site-directed mutagenesis at the Mg2+-liganding residue Asp-246. The mutant proteins exhibited approximately 20-fold reduced ATPase activity but normal strand exchange activity. Structurally, the main chain carbonyl of the conserved catalytic residue Glu-151 is hydrogen bonded with one of the magnesium-liganding water molecules. Changes in the secondary magnesium site may therefore induce conformational changes around this catalytic glutamate and affect the ATPase activity without significantly altering the stability of the extended recombinase filament. Asp-246 is somewhat conserved among archaeal and eukaryal homologues, implying some homologues may share this allosteric site for ATPase function.

Calcium stiffens archaeal Rad51 recombinase from Methanococcus voltae for homologous recombination.

Archaeal RadA or Rad51 recombinases are close homologues of eukaryal Rad51 and DMC1. These and bacterial RecA orthologues play a key role in DNA repair by forming helical nucleoprotein filaments in which a hallmark strand exchange reaction between homologous DNA substrates occurs. Recent studies have discovered the stimulatory role by calcium on human and yeast recombinases. Here we report that the strand exchange activity but not the ATPase activity of an archaeal RadA/Rad51 recombinase from Methanococcus voltae (MvRadA) is also subject to calcium stimulation. Crystallized MvRadA filaments in the presence of CaCl(2) resemble that of the recently reported ATPase active form in the presence of an activating dose of KCl. At the ATPase center, one Ca(2+) ion takes the place of two K(+) ions in the K(+)-bound form. The terminal phosphate of the nonhydrolyzable ATP analogue is in a staggered conformation in the Ca(2+)-bound form. In comparison, an eclipsed conformation was seen in the K(+)-bound form. Despite the changes in the ATPase center, both forms harbor largely ordered L2 regions in essentially identical conformations. These data suggest a unified stimulation mechanism by potassium and calcium because of the existence of a conserved ATPase center promiscuous in binding cations.

The Rad51/RadA N-terminal domain activates nucleoprotein filament ATPase activity.

Proteins in the RecA/RadA/Rad51 family form helical filaments on DNA that function in homologous recombination. While these proteins all have the same highly conserved ATP binding core, the RadA/Rad51 proteins have an N-terminal domain that shows no homology with the C-terminal domain found in RecA. Both the Rad51 N-terminal and RecA C-terminal domains have been shown to bind DNA, but no role for these domains has been established. We show that RadA filaments can be trapped in either an inactive or active conformation with respect to the ATPase and that activation involves a large rotation of the subunit aided by the N-terminal domain. The G103E mutation within the yeast Rad51 N-terminal domain inactivates the filament by failing to make proper contacts between the N-terminal domain and the core. These results show that the N-terminal domains play a regulatory role in filament activation and highlight the modular architecture of the recombination proteins.

Asp302 determines potassium dependence of a RadA recombinase from Methanococcus voltae.

Archaeal RadA/Rad51 are close homologues of eukaryal Rad51/DMC1. Such recombinases, as well as their bacterial RecA orthologues, form helical nucleoprotein filaments in which a hallmark strand exchange reaction occurs between homologous DNA substrates. Our recent ATPase and structure studies on RadA recombinase from Methanococcus voltae have suggested that not only magnesium but also potassium ions are absorbed at the ATPase center. Potassium, but not sodium, stimulates the ATP hydrolysis reaction with an apparent dissociation constant of approximately 40 mM. The minimal inhibitory effect by 40 mM NaCl further suggests that the protein does not have adequate affinity for sodium. The wild-type protein's strand exchange activity is also stimulated by potassium with an apparent dissociation constant of approximately 35 mM. We made site-directed mutations at the potassium-contacting residues Glu151 and Asp302. The mutant proteins are expectedly defective in promoting ATP hydrolysis. Similar potassium preference in strand exchange is observed for the E151D and E151K proteins. The D302K protein, however, shows comparable strand exchange efficiencies in the presence of either potassium or sodium. Crystallized E151D filaments reveal a potassium-dependent conformational change similar to what has previously been observed with the wild-type protein. We interpret these data as suggesting that both ATP hydrolysis and DNA strand exchange requires accessibility to an "active" conformation similar to the crystallized ATPase-active form in the presence of ATP, Mg2+ and K+.

Crystal structure of Methanococcus voltae RadA in complex with ADP: hydrolysis-induced conformational change.

Members of a superfamily of RecA-like recombinases facilitate a central strand exchange reaction in the DNA repair process. Archaeal RadA and Rad51 and eukaryal Rad51 and meiosis-specific DMC1 form a closely related group of recombinases distinct from bacterial RecA. Nevertheless, all such recombinases share a conserved core domain which carries the ATPase site and putative DNA-binding sites. Here we present the crystal structure of an archaeal RadA from Methanococcus voltae (MvRadA) in complex with ADP and Mg2+ at 2.1 A resolution. The crystallized RadA-ADP filament has an extended helical pitch similar to those of previously determined structures in the presence of nonhydrolyzable ATP analogue AMP-PNP. Structural comparison reveals two recurrent conformations with an extensive allosteric effect spanning the ATPase site and the putative DNA-binding L2 region. Varied conformations of the L2 region also imply a dynamic nature of recombinase-bound DNA.

Crystal structure of an ATPase-active form of Rad51 homolog from Methanococcus voltae. Insights into potassium dependence.

Homologous gene recombination is crucial for the repair of DNA. A superfamily of recombinases facilitate a central strand exchange reaction in the repair process. This reaction is initiated by coating single-stranded DNA (ssDNA) with recombinases in the presence of ATP and Mg(2+) co-factors to form helical nucleoprotein filaments with elevated ATPase and strand invasion activities. At the amino acid sequence level, archaeal RadA and Rad51 and eukaryal Rad51 and meiosis-specific DMC1 form a closely related group of recombinases distinct from bacterial RecA. Unlike the extensively studied Escherichia coli RecA (EcRecA), increasing evidences on yeast and human recombinases imply that their optimal activities are dependent on the presence of a monovalent cation, particularly potassium. Here we present the finding that archaeal RadA from Methanococcus voltae (MvRadA) is a stringent potassium-dependent ATPase, and the crystal structure of this protein in complex with the non-hydrolyzable ATP analog adenosine 5'-(beta,gamma-iminotriphosphate), Mg(2+), and K(+) at 2.4 A resolution. Potassium triggered an in situ conformational change in the ssDNA-binding L2 region concerted with incorporation of two potassium ions at the ATPase site in the RadA crystals preformed in K(+)-free medium. Both potassium ions were observed in contact with the gamma-phosphate of the ATP analog, implying a direct role by the monovalent cations in stimulating the ATPase activity. Cross-talk between the ATPase site and the ssDNA-binding L2 region visualized in the MvRadA structure provides an explanation to the co-factor-induced allosteric effect on RecA-like recombinases.

Crystal structure of archaeal recombinase RADA: a snapshot of its extended conformation.

Homologous recombination of DNA plays crucial roles in repairing severe DNA damage and in generating genetic diversity. The process is facilitated by a superfamily of recombinases: bacterial RecA, archaeal RadA and Rad51, and eukaryal Rad51 and DMC1. These recombinases share a common ATP-dependent filamentous quaternary structure for binding DNA and facilitating strand exchange. We have determined the crystal structure of Methanococcus voltae RadA in complex with the ATP analog AMP-PNP at 2.0 A resolution. The RadA filament is a 106.7 A pitch helix with six subunits per turn. The DNA binding loops L1 and L2 are located in close proximity to the filament axis. The ATP analog is buried between two RadA subunits, a feature similar to that of the active filament of Escherichia coli RecA revealed by electron microscopy. The disposition of the N-terminal domain suggests a role of the Helix-hairpin-Helix motif in binding double-stranded DNA.

[Nucleotide sequence and protein sequence analysis of GL-7-ACA acylase from Pseudomonas sp. 130].

The nucleotide sequence and N-, C-terminal amino acid sequences of alpha,beta-subunit of glutaryl 7-ACA acylase C130 from Pseudomonas sp. 130 were determined. The alignment of the acylase C130 with the other acylases shows that it has high homology with the acylases from Pseudomonas sp. GK16 and C427, but low homology with the others. There is large difference in the N-terminal of alpha-subunit, while the N-terminal of beta-subunit has significant conservation.