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1.
The nuclease NurA is present in all known thermophilic archaea and has been implicated to facilitate efficient DNA double-strand
break end processing in Mre11/Rad50-mediated homologous recombinational repair. To understand the structural and functional
relationship of this enzyme, we constructed five site-directed mutants of NurA from Sulfolobus tokodaii (StoNurA), D56A, E114A, D131A, Y291A, and H299A, at the conserved motifs, and four terminal deletion mutants, StoNurAΔN (19–331),
StoNurAΔNΔC (19–303), StoNurAΔC (1–281), and StoNurAΔC (1–303), and characterized the proteins biochemically. We found that
mutation at the acidic residue, D56, E114, D131, or at the basic residue, H299, abolishes the nuclease activity, while mutation
at the aromatic residue Y291 only impairs the activity. Interestingly, by chemical cross-linking assay, we found that the
mutant Y291A is unable to form stable dimer. Additionally, we demonstrated that deletion of the C-terminal amino acid residues
304–331 of StoNurA results in loss of the physical and functional interaction with the single-stranded DNA-binding protein
(StoSSB). These results established that the C-terminal conserved aromatic residue Y291 is involved in dimer formation and
the C-terminal residues 304–331 of NurA are involved in the interaction with single-stranded DNA-binding protein. 相似文献
2.
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. 相似文献
3.
Recent crystallographic resolution of ?29 DNA polymerase complexes with ssDNA at its 3′-5′ exonuclease active site has allowed the identification of residues Pro129 and Tyr148 as putative ssDNA ligands, the latter being conserved in the Kx 2h motif of proofreading family B DNA polymerases. Single substitution of ?29 DNA polymerase residue Tyr148 to Ala rendered an enzyme with a reduced capacity to stabilize the binding of the primer terminus at the 3′-5′ exonuclease active site, not having a direct role in the catalysis of the reaction. Analysis of the 3′-5′ exonuclease on primer/template structures showed a critical role for residue Tyr148 in the proofreading of DNA polymerisation errors. In addition, Tyr148 is not involved in coupling polymerisation to strand displacement in contrast to the catalytic residues responsible for the exonuclease reaction, its role being restricted to stabilisation of the frayed 3′ terminus at the exonuclease active site. Altogether, the results lead us to extend the consensus sequence of the above motif of proofreading family B DNA polymerases into Kx 2hx A. The different solutions adopted by proofreading DNA polymerases to stack the 3′ terminus at the exonuclease site are discussed. In addition, the results obtained with mutants at ?29 DNA polymerase residue Pro129 allow us to rule out a functional role as ssDNA ligand for this residue. 相似文献
4.
RecJ exonuclease plays crucial roles in several DNA repair and recombination pathways, and its ubiquity in bacterial species points to its ancient origin and vital cellular function. RecJ exonuclease from Haemophilus influenzae is a 575-amino-acid protein that harbors the characteristic motifs conserved among RecJ homologs. The purified protein exhibits a processive 5′-3′ single-stranded-DNA-specific exonuclease activity. The exonuclease activity of H. influenzae RecJ (HiRecJ) was supported by Mg 2 + or Mn 2 + and inhibited by Cd 2 +, suggesting a different mode of metal binding in HiRecJ as compared to Escherichia coli RecJ (EcoRecJ). Site-directed mutagenesis of highly conserved residues in HiRecJ abolished enzymatic activity. Interestingly, substitution of alanine for aspartate 77 resulted in a catalytically inactive enzyme that bound to DNA with a significantly higher affinity as compared to the wild-type enzyme. Noticeably, steady-state kinetic studies showed that H. influenzae single-stranded DNA-binding protein (HiSSB) increased the affinity of HiRecJ for single-stranded DNA and stimulated its exonuclease activity. HiSSB, whose C-terminal tail had been deleted, failed to enhance RecJ exonuclease activity. More importantly, HiRecJ was found to directly associate with its cognate single-stranded DNA-binding protein (SSB), as demonstrated by various in vitro assays. Interaction studies carried out with the truncated variants of HiRecJ and HiSSB revealed that the two proteins interact via the C-terminus of SSB protein and the core-catalytic domain of RecJ. Taken together, these results emphasize direct interaction between RecJ and SSB, which confers functional cooperativity to these two proteins. In addition, these results implicate SSB as being involved in the recruitment of RecJ to DNA and provide insights into the interplay between these proteins in repair and recombination pathways. 相似文献
5.
The nuclease NurA and the ATPase HerA are present in all known thermophilic archaea and cooperate with the highly conserved MRE11/RAD50 proteins to facilitate efficient DNA double-strand break end processing during homologous recombinational repair. However, contradictory results have been reported on the exact activities and mutual dependence of these two enzymes. To understand the functional relationship between these two enzymes we deeply characterized Sulfolobus solfataricus NurA and HerA proteins. We found that NurA is endowed with exo- and endonuclease activities on various DNA substrates, including linear (single-stranded and double stranded) as well as circular molecules (single stranded and supercoiled double-stranded). All these activities are not strictly dependent on the presence of HerA, require divalent ions (preferably Mn 2+), and are inhibited by the presence of ATP. The endo- and exonculease activities have distinct requirements: whereas the exonuclease activity on linear DNA fragments is stimulated by HerA and depends on the catalytic D58 residue, the endonuclease activity on circular double-stranded DNA is HerA-independent and is not affected by the D58A mutation. On the basis of our results we propose a mechanism of action of NurA/HerA complex during DNA end processing. 相似文献
6.
We isolated and characterized a new nuclease (NurA) exhibiting both single-stranded endonuclease activity and 5′–3′ exonuclease activity on single-stranded and double-stranded DNA from the hyperthermophilic archaeon Sulfolobus acidocaldarius. Nuclease homologs are detected in all thermophilic archaea and, in most species, the nurA gene is organized in an operon-like structure with rad50 and mre11 archaeal homologs. This nuclease might thus act in concert with Rad50 and Mre11 proteins in archaeal recombination/repair. To our knowledge, this is the first report of a 5′–3′ nuclease potentially associated with Rad50 and Mre11-like proteins that may lead to the processing of double-stranded breaks in 3′ single-stranded tails. 相似文献
7.
The Mre11/Rad50 complex has been implicated in the early steps of DNA double-strand break (DSB) repair through homologous recombination in several organisms. However, the enzymatic properties of this complex are incompatible with the generation of 3' single-stranded DNA for recombinase loading and strand exchange. In thermophilic archaea, the Mre11 and Rad50 genes cluster in an operon with genes encoding a helicase, HerA, and a 5' to 3' exonuclease, NurA, suggesting a common function. Here we show that purified Mre11 and Rad50 from Pyrococcus furiosus act cooperatively with HerA and NurA to resect the 5' strand at a DNA end under physiological conditions in vitro. The 3' single-stranded DNA generated by these enzymes can be utilized by the archaeal RecA homolog RadA to catalyze strand exchange. This work elucidates how the conserved Mre11/Rad50 complex promotes DNA end resection in archaea and may serve as a model for DSB processing in eukaryotes. 相似文献
8.
PriB is a basic 10-kDa protein that acts as a facilitator in PriA-dependent replication restart in Escherichia coli. PriB has an OB-fold dimer structure and exhibits single-stranded DNA (ssDNA)-binding activities similar to single-stranded binding protein (SSB). In this study, we examined PriB's interaction with ssDNA (oligo-dT35, -dT15, and -dT7) using heteronuclear NMR analysis. Interestingly, 1H or 15N chemical shift changes of the PriB main-chain showed two distinct modes using oligo-dT35. The chemical shift perturbation sites in the primary mode were consistent with the main contact site in PriB–ssDNA, which was previously determined by crystal structure analysis. The results also suggested that approximately 8 nt in ssDNA was the main contact site to PriB. In the secondary mode, residues in the α-helix region (His57–Ser65) and in β4–loop3–β5 were mainly perturbed. On the other hand, we examined the state of ssDNA by FRET using 5′-Cy3- and 3′-Cy5-modified oligo-dT35. As the PriB concentration increased, two-step saturation curves were observed in the FRET assay, suggesting a compact structure of ssDNA. Moreover, we confirmed two-step PriB binding to oligo-dT35 using EMSA. The pH dependence of FRET suggested contribution of the His residues. Therefore, we prepared His mutants of PriB and found that His64 in the α-helix region contributed to the second interaction between PriB and ssDNA using FRET and EMSA. Thus, from a structural standpoint, we suggested the role of His64 on the compactness of the PriB–ssDNA complex and on the positive cooperativity of PriB. 相似文献
9.
We have developed a method that allows quantitative amplification of single-stranded DNA (QAOS) in a sample that is primarily double-stranded DNA (dsDNA). Single-stranded DNA (ssDNA) is first captured by annealing a tagging primer at low temperature. Primer extension follows to create a novel, ssDNA-dependent, tagged molecule that can be detected by PCR. Using QAOS levels of between 0.2 and 100% ssDNA can be accurately quantified. We have used QAOS to characterise ssDNA levels at three loci near the right telomere of chromosome V in budding yeast cdc13-1 mutants. Our results confirm and extend previous studies which demonstrate that when Cdc13p, a telomere-binding protein, is disabled, loci close to the telomere become single stranded whereas centromere proximal sequences do not. In contrast to an earlier model, our new results are consistent with a model in which a RAD24-dependent, 5′ to 3′ exonuclease moves from the telomere toward the centromere in cdc13-1 mutants. QAOS has been adapted, using degenerate tagging primers, to preferentially amplify all ssDNA sequences within samples that are primarily dsDNA. This approach may be useful for identifying ssDNA sequences associated with physiological or pathological states in other organisms. 相似文献
11.
DNA double-strand breaks can be repaired by homologous recombination, during which the DNA ends are long-range resected by helicase–nuclease systems to generate 3′ single strand tails. In archaea, this requires the Mre11–Rad50 complex and the ATP-dependent helicase–nuclease complex HerA–NurA. We report the cryo-EM structure of Sulfolobus solfataricus HerA–NurA at 7.4 Å resolution and present the pseudo-atomic model of the complex. HerA forms an ASCE hexamer that tightly interacts with a NurA dimer, with each NurA protomer binding three adjacent HerA HAS domains. Entry to NurA’s nuclease active sites requires dsDNA to pass through a 23 Å wide channel in the HerA hexamer. The structure suggests that HerA is a dsDNA translocase that feeds DNA into the NurA nuclease sites. 相似文献
13.
Exonuclease X is a 3′-5′ distributive exonuclease that functions in DNA recombination and repair. It undergoes multiple rounds of binding, hydrolysis, and release to degrade long substrate molecules and thus is very inefficient. In order to identify a cofactor that elevates the excision activity of ExoX, we screened many proteins involved in repair and recombination. We observed that MutL greatly promoted the exonuclease activity of ExoX, and then verified the interaction between MutL and ExoX using SPR and Far-Western analysis. This promotion is independent of ATP and the DNA-binding activity of MutL. We constructed two deletion mutants to analyze this interaction and its regulation of ExoX activity, and found that this functional interaction with ExoX is mainly due to ionic interactions with the N-terminus of MutL. This adds a new role to MutL and gives a clue to MutL’s possible regulation on other DnaQ family exonuclease members. 相似文献
14.
Deamination of adenine can occur spontaneously under physiological conditions, and is enhanced by exposure of DNA to ionizing radiation, UV light, nitrous acid, or heat, generating the highly mutagenic lesion of deoxyinosine in DNA. Such DNA lesions tends to generate A:T to G:C transition mutations if unrepaired. In Escherichia coli, deoxyinosine is primarily removed through a repair pathway initiated by endonuclease V (endo V). In this study, we compared the repair of three mutagenic deoxyinosine lesions of A-I, G-I, and T-I using E. coli cell-free extracts as well as reconstituted protein system. We found that 3′-5′ exonuclease activity of DNA polymerase I (pol I) was very important for processing all deoxyinosine lesions. To understand the nature of pol I in removing damaged nucleotides, we systemically analyzed its proofreading to 12 possible mismatches 3′-penultimate of a nick, a configuration that represents a repair intermediate generated by endo V. The results showed all mismatches as well as deoxyinosine at the 3′ penultimate site were corrected with similar efficiency. This study strongly supports for the idea that the 3′-5′ exonuclease activity of E. coli pol I is the primary exonuclease activity for removing 3′-penultimate deoxyinosines derived from endo V nicking reaction. 相似文献
15.
Nucleases play important roles in all DNA transactions, including replication, repair, and recombination. Many different nucleases from bacterial and eukaryotic organisms have been identified and functionally characterized. However, our knowledge about the nucleases from Archaea, the third domain of life, is still limited. We searched for 3′–5′ exonuclease activity in the hyperthermophilic archaeon, Pyrococcus furiosus, and identified a protein with the target activity. The purified protein, encoded by PF2046, is composed of 229 amino acids with a molecular weight of 25,596, and displayed single-strand specific 3′–5′ exonuclease activity. The protein, designated as PfuExo I, forms a stable trimeric complex in solution and excises the DNA at every two nucleotides from the 3′ to 5′ direction. The amino acid sequence of this protein is conserved only in Thermococci, one of the hyperthermophilic classes in the Euryarchaeota subdomain in Archaea. The newly discovered exonuclease lacks similarity to any other proteins with known function, including hitherto reported 3′–5′ exonucleases. This novel nuclease may be involved in a DNA repair pathway conserved in the living organisms as a specific member for some hyperthermophilic archaea. 相似文献
16.
The RecJ exonuclease from Escherichia coli degrades single-stranded DNA (ssDNA) in the 5′–3′ direction and participates in homologous recombination and mismatch repair. The experiments described here address RecJ's substrate requirements and reaction products. RecJ complexes on a variety of 5′ single-strand tailed substrates were analyzed by electrophoretic mobility shift in the absence of Mg 2+ ion required for substrate degradation. RecJ required single-stranded tails of 7 nt or greater for robust binding; addition of Mg 2+ confirmed that substrates with 5′ tails of 6 nt or less were poor substrates for RecJ exonuclease. RecJ is a processive exonuclease, degrading ~1000 nt after a single binding event to single-strand DNA, and releases mononucleotide products. RecJ is capable of degrading a single-stranded tail up to a double-stranded junction, although products in such reactions were heterogeneous and RecJ showed a limited ability to penetrate the duplex region. RecJ exonuclease was equally potent on 5′ phosphorylated and unphosphorylated ends. Finally, DNA binding and nuclease activity of RecJ was specifically enhanced by the pre-addition of ssDNA-binding protein and we propose that this specific interaction may aid recruitment of RecJ. 相似文献
17.
Streptomyces coelicolor A3(2) produces several intra and extracellular enzymes with deoxyribonuclease activities. The examined N-terminal amino acid sequence of one of extracellular DNAases (TVTSVNVNGLL) and database search on S. coelicolor genome showed a significant homology to the putative secreted exodeoxyribonuclease. The corresponding gene ( exoSc) was amplified, cloned, expressed in Escherichia coli, purified to homogeneity and characterized. Exonuclease recExoSc degraded chromosomal, linear dsDNA with 3′-overhang ends, linear ssDNA and did not digest linear dsDNA with blunt ends, supercoiled plasmid ds nor ssDNA. The substrate specificity of recExoSc was in the order of dsDNA > ssDNA > 3′-dAMP. The purified recExoSc was not a metalloprotein and exhibited neither phosphodiesterase nor RNase activity. It acted as 3′-phosphomonoesterase only at 3′-dAMP as a substrate. The optimal temperature for its activity was 57 °C in Tris–HCl buffer at optimal pH = 7.5 for either ssDNA or dsDNA substrates. It required a divalent cation (Mg 2+, Co 2+, Ca 2+) and its activity was strongly inhibited in the presence of Zn 2+, Hg 2+, chelating agents or iodoacetate. 相似文献
18.
The single-stranded DNA (ssDNA)-binding protein replication protein A (RPA) is essential for both DNA replication and recombination. Chromatin immunoprecipitation techniques were used to visualize the kinetics and extent of RPA binding following induction of a double-strand break (DSB) and during its repair by homologous recombination in yeast. RPA assembles at the HO endonuclease-cut MAT locus simultaneously with the appearance of the DSB, and binding spreads away from the DSB as 5′ to 3′ exonuclease activity creates more ssDNA. RPA binding precedes binding of the Rad51 recombination protein. The extent of RPA binding is greater when Rad51 is absent, supporting the idea that Rad51 displaces RPA from ssDNA. RPA plays an important role during RAD51-mediated strand invasion of the MAT ssDNA into the donor sequence HML. The replication-proficient but recombination-defective rfa1-t11 (K45E) mutation in the large subunit of RPA is normal in facilitating Rad51 filament formation on ssDNA, but is unable to achieve synapsis between MAT and HML. Thus, RPA appears to play a role in strand invasion as well as in facilitating Rad51 binding to ssDNA, possibly by stabilizing the displaced ssDNA. 相似文献
19.
The fidelity of DNA synthesis by DNA polymerase is significantly increased by a mechanism of proofreading that is performed at the exonuclease active site separate from the polymerase active site. Thus, the transition of DNA between the two active sites is an important activity of DNA polymerase. Here, based on our proposed model, the rates of DNA transition between the two active sites are theoretically studied. With the relevant parameters, which are determined from the available crystal structure and other experimental data, the calculated transfer rate of correctly base-paired DNA from the polymerase to exonuclease sites and the transfer rate after incorporation of a mismatched base are in good agreement with the available experimental data. The transfer rates in the presence of two and three mismatched bases are also consistent with the previous experimental data. In addition, the calculated transfer rate from the exonuclease to polymerase sites has a large value even with the high binding affinity of 3′-5′ ssDNA for the exonuclease site, which is also consistent with the available experimental value. Moreover, we also give some predictive results for the transfer rate of DNA containing only A:T base pairs and that of DNA containing only G:C base pairs. 相似文献
20.
(2′,3′- O-Isopropylidene-5′-uridyl) 4-(2,3,4,6-tetra- O-acetyl-β-d-glycopyranosyl)allophanates were obtained in the reactions of 2′,3′- O-isopropylidene-uridine and O-peracetylated β-d-gluco-, galacto- and xylopyranosylamines, and OCNCOCl. 2,3,4,6-Tetra- O-acetyl-β-d-glucopyranosyl isocyanate and N-(2′,3′- O-isopropylidene-5′-uridyl)urea gave 1-(2,3,4,6-tetra- O-acetyl-β-d-glucopyranosyl)-5-(2′,3′- O-isopropylidene-5′-uridyl)biuret. Deprotection of the β-d- gluco configured allophanate and biuret was carried out by standard methods. 相似文献
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