Biochemical characterization and mutational analysis of the NurA protein from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5.

Archaeal NurA protein plays a key role in producing 3'-single stranded DNA used for homologous recombination repair, together with HerA, Mre11, and Rad50. Herein, we describe biochemical characteristics and roles of key amino acid residues of the NurA protein from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 (Tba-NurA). Tba-NurA possesses 5'-3' exonuclease activity for degrading DNA, displaying maximum efficiency at 45 °C-65 °C and at pH 8.0 in the presence of Mn2+. The thermostable Tba-NurA also possesses endonuclease activity capable of nicking plasmid DNA and circular ssDNA. Mutational data demonstrate that residue D49 of Tba-NurA is essential for exonuclease activity and is involved in binding ssDNA since the D49A mutant lacked exonuclease activity and reduced ssDNA binding. The R96A and R129A mutants had no detectable dsDNA binding, suggesting that residues R96 and R129 are important for binding dsDNA. The abolished degradation activity and reduced dsDNA binding of the D120A mutant suggest that residue D120 is essential for degradation activity and dsDNA binding. Additionally, residues Y392 and H400 are important for exonuclease activity since these mutations resulted in exonuclease activity loss. To our knowledge, it is the first report on biochemical characterization and mutational analysis of the NurA protein from Thermococcus.

Flexible and excellent electromagnetic interference shielding film with porous alternating PVA-derived carbon and graphene layers.

Recently, the design of graphene-based films with elaborately controlled microstructures and optimized electromagnetic interference shielding (EMI) properties can effectively improve EM energy attenuation and conversion. Herein, inspired by the structure of multi-layer steamed bread, an alternating multilayered structure with polyvinyl alcohol (PVA)-derived carbon layers and graphene/electrospun carbon nanofibers layers was designed through alternating vacuum-assisted filtration method. The composite film exhibited favorable impedance matching, abundant loss mechanism, and excellent EMI shielding ability, resulting in absorption dominated shielding characteristic. Thus, the resultant 7-layer alternating composite films with a thickness of 160 µm achieved an EMI shielding effectiveness (EMI SE) of up to 80 dB in the X-band. Specially, finite element analysis was applied to demonstrate the importance of seven-layer film alternations and detailed analysis of electromagnetic shielding mechanisms. Taken together, this effort opens a creative avenue for designing and constructing flexible composite films with excellent EMI shielding performance.

Thermostable DNA ligases from hyperthermophiles in biotechnology.

DNA ligase is an important enzyme ubiquitous in all three kingdoms of life that can ligate DNA strands, thus playing essential roles in DNA replication, repair and recombination in vivo. In vitro, DNA ligase is also used in biotechnological applications requiring in DNA manipulation, including molecular cloning, mutation detection, DNA assembly, DNA sequencing, and other aspects. Thermophilic and thermostable enzymes from hyperthermophiles that thrive in the high-temperature (above 80°C) environments have provided an important pool of useful enzymes as biotechnological reagents. Similar to other organisms, each hyperthermophile harbors at least one DNA ligase. In this review, we summarize recent progress on structural and biochemical properties of thermostable DNA ligases from hyperthermophiles, focusing on similarities and differences between DNA ligases from hyperthermophilic bacteria and archaea, and between these thermostable DNA ligases and non-thermostable homologs. Additionally, altered thermostable DNA ligases are discussed. Possessing improved fidelity or thermostability compared to the wild-type enzymes, they could be potential DNA ligases for biotechnology in the future. Importantly, we also describe current applications of thermostable DNA ligases from hyperthermophiles in biotechnology.

[ST266 inhibits neointimal hyperplasia after arterial balloon injury in rats].

Objective    To examine the effect of Human Amnion-Derived Multipotent Progenitor (AMP) cells and their novel ST266 secretome on neointimal hyperplasia after arterial balloon injury in rats.Material and Methods    Sprague-Dawley male rats were randomly divided into four groups (n=7): Control (PBS) group, systemic ST266 group, systemic AMP group and local AMP implant group. Neointimal hyperplasia was induced in the iliac using a 2F Fogarty embolectomy catheter. After surgery, the rats in the ST266 group were treated with 0.1, 0.5, or 1ml ST266 iv daily. In the systemic AMP groups, a single dose (SD) of 0.5 ×106 or 1×106 AMP cells was injected via the inferior vena cava after arterial balloon injury. In local AMP implant groups, 1×106, 5×106, or 20×106 AMP cells were implanted in 300 µl Matrigel (Mtgl) around the iliac artery after balloon injury. The iliac arteries were removed for histologic analysis at 28 days after the surgery. Re-endothelialization index was measured at 10 days after balloon injury.Results    ST266 (1 ml) group had a lower level of the Neointima / Neointima+Media ratio (N / N+M) 0.3±0.1 vs 0.5±0.1, p=0.004) and luminal stenosis (LS) percentage (18.2±1.9 % vs 39.2±5.8 %, p=0.008) compared with the control group. Single-dose AMP (1×106) decreased LS compared to the control group (19.5±5.4 % vs 39.2±5.8 %, p=0.033). Significant reduction in N / N+M were found between implanted AMPs (20×106) and the control group (0.4±0.1 vs 0.5±0.1, p=0.003) and the Mtgl-only group (0.5±0.1, p=0.007). Implanted AMPs (20×106) decreased the LS compared with both the control (39.2±5.8 %, p=0.001) and Mtgl-only group (37.5±8.6 %, p=0.016). ST266 (1 ml) significantly increased the re-endothelialization index compared to the control (0.4±0.1 vs 0.1±0.1, p=0.002).Conclusion    ST266 and AMP cells reduce neointimal formation and increase the re-endothelialization index after arterial balloon injury. ST266 is potentially a novel, therapeutic agent to prevent vascular restenosis in human.

Archaeal DNA alkylation repair conducted by DNA glycosylase and methyltransferase.

Alkylated bases in DNA created in the presence of endogenous and exogenous alkylating agents are either cytotoxic or mutagenic, or both to a cell. Currently, cells have evolved several strategies for repairing alkylated base. One strategy is a base excision repair process triggered by a specific DNA glycosylase that is used for the repair of the cytotoxic 3-methyladenine. Additionally, the cytotoxic and mutagenic O6-methylguanine (O6-meG) is corrected by O6-methylguanine methyltransferase (MGMT) via directly transferring the methyl group in the lesion to a specific cysteine in this protein. Furthermore, oxidative DNA demethylation catalyzed by DNA dioxygenase is utilized for repairing the cytotoxic 3-methylcytosine (3-meC) and 1-methyladenine (1-meA) in a direct reversal manner. As the third domain of life, Archaea possess 3-methyladenine DNA glycosylase II (AlkA) and MGMT, but no DNA dioxygenase homologue responsible for oxidative demethylation. Herein, we summarize recent progress in structural and biochemical properties of archaeal AlkA and MGMT to gain a better understanding of archaeal DNA alkylation repair, focusing on similarities and differences between the proteins from different archaeal species and between these archaeal proteins and their bacterial and eukaryotic relatives. To our knowledge, it is the first review on archaeal DNA alkylation repair conducted by DNA glycosylase and methyltransferase. KEY POINTS: • Archaeal MGMT plays an essential role in the repair of O 6 -meG • Archaeal AlkA can repair 3-meC and 1-meA.

Biochemical characterization and mutational studies of endonuclease Q from the hyperthermophilic euryarchaeon Thermococcus gammatolerans.

Endonuclease Q (EndoQ) can effectively cleave DNA containing deaminated base(s), thus providing a potential pathway for repair of deaminated DNA. EndoQ is ubiquitous in some Archaea, especially in Thermococcales, and in a small group of bacteria. Herein, we report biochemical characteristics of EndoQ from the hyperthermophilic euryarchaeon Thermococcus gammatolerans (Tga-EndoQ) and the roles of its six conserved residues in DNA cleavage. The enzyme can cleave uracil-, hypoxanthine-, and AP (apurinic/apyrimidinic) site-containing DNA with varied efficiencies at high temperature, among which uracil-containing DNA is its most preferable substrate. Additionally, the enzyme displays maximum cleavage efficiency at above 70 oC and pH 7.0 ∼ 8.0. Furthermore, Tga-EndoQ still retains 85% activity after heated at 100 oC for 2 hrs, suggesting that the enzyme is extremely thermostable. Moreover, the Tga-EndoQ activity is independent of a divalent ion and NaCl. Mutational data demonstrate that residues E167 and H195 in Tga-EndoQ are essential for catalysis since the E167A and H195A mutants completely abolish the cleavage activity. Besides, residues S18 and R204 in Tga-EndoQ are involved in catalysis due to the reduced activities observed for the S18A and R204A mutants. Overall, our work has augmented biochemical function of archaeal EndoQ and provided insight into its catalytic mechanism.

Biochemical and mutational studies of an endonuclease V from the hyperthermophilic crenarchaeon Sulfolobus islandicus REY15A.

Endonuclease V (EndoV), which is widespread in bacteria, eukarya and Archaea, can cleave hypoxanthine (Hx)-containing DNA or RNA strand, and play an essential role in Hx repair. However, our understanding on archaeal EndoV's function remains incomplete. The model archaeon Sulfolobus islandicus REY15A encodes a putative EndoV protein (Sis-EndoV). Herein, we probed the biochemical characteristics of Sis-EndoV and dissected the roles of its seven conserved residues. Our biochemical data demonstrate that Sis-EndoV displays maximum cleavage efficiency at above 60 °C and at pH 7.0-9.0, and the enzyme activity is dependent on a divalent metal ion, among which Mg2+ is optimal. Importantly, we first measured the activation energy for cleaving Hx-containing ssDNA by Sis-EndoV to be 9.6 ± 0.8 kcal/mol by kinetic analyses, suggesting that chemical catalysis might be a rate-limiting step for catalysis. Mutational analyses show that residue D38 in Sis-EndoV is essential for catalysis, but has no role in DNA binding. Furthermore, we first revealed that residues Y41 and D189 in Sis-EndoV are involved in both DNA cleavage and DNA binding, but residues F77, H103, K156 and F161 are only responsible for DNA binding.

Biochemical characterization and mechanistic insight of the family IV uracil DNA glycosylase from Sulfolobus islandicus REY15A.

Uracil DNA glycosylase (UDG) can remove uracil from DNA, thus playing an essential role in maintaining genomic stability. Family IV UDG members are mostly widespread in hyperthermophilic Archaea and bacteria. In this work, we characterized the family IV UDG from the hyperthermophilic crenarchaeon Sulfolobus islandicus REY15A (Sis-UDGIV) biochemically, and dissected the roles of nine conserved residues in uracil excision by mutational analyses. Biochemical data demonstrate that Sis-UDGIV displays maximum efficiency for uracil excision at 50 °C ~ 70 °C and at pH 7.0-9.0. Additionally, the enzyme has displays a weak activity without a divalent metal ion, but maximum activity with Mg2+. Our mutational analyses show that residues E48 and F55 in Sis-UDGIV are essential for uracil removal, and residues E48, F55, R87, R92 and K146 are responsible for binding DNA. Importantly, we systemically revealed the roles of four conserved cysteine residues C14, C17, C86 and C102 in Sis-UDGIV that are required for being ligands of FeS cluster in maintaining the overall protein conformation and stability by circular dichroism analyses. Overall, our work has provided insights into biochemical function and DNA-binding specificity of archaeal family IV UDGs.

Single-cell transcriptomic, transcriptomic, and metabolomic characterization of human atherosclerosis.

Background: Atherosclerosis is the main cause of many cardiovascular and cerebrovascular diseases (CVDs), and gaining a deeper understanding of the intercellular connections and key central genes which mediate formation of atherosclerotic plaques is required. Methods: We performed a comprehensive bioinformatics analysis of differential genetic screening, Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway annotation, protein-protein interactions (PPIs), pseudo-timing, intercellular communication, transcription factors on carotid single-cell sequencing data, and aortic bulk transcriptome and metabolomic data. Results: Ten cell types were identified in the data: T cells, monocytes, smooth muscle cells, endothelial cells, B cells, fibroblasts, plasma cells, mast cells, dendritic cells, and natural killer cells. Endothelial, fibroblast, macrophage, and smooth muscle cell subtype differentiation trajectories, interaction networks, and important transcription factors were characterized in detail. Finally, using this information combined with transcriptome and metabolome analyses, we found the key genes and signaling pathways of atherosclerosis, especially the advanced glycation end products and receptor for advanced glycation end products signaling pathway (AGE-RAGE signaling pathway) in diabetic complications, linked the differential metabolites with fibroblasts and atherosclerosis and contributed to it in patients with diabetes. Conclusions: Collectively, this study provides key genes, signaling pathways, cellular communication, and transcription factors among endothelial cells, fibroblasts, macrophages, and smooth muscle cells for the study of atherosclerotic plaques, and provides a basis for the diagnosis and treatment of atherosclerosis-like sclerosis.

A novel Family V uracil DNA glycosylase from Sulfolobus islandicus REY15A.

Uracil DNA glycosylase (UDG) can excise uracil from DNA, thus playing an essential role in counteracting mutations. The genome of the hyperthermophilic crenarchaeon Sulfolobus islandicus REY15A encodes one putative Family V UDG (Sis-UDGV). Herein, we provide evidence that Sis-UDGV is a bi-functional glycosylase that can not only excise uracil from DNA, but cleave the generated apurinic/apyrimidinic (AP) site, which differs from other reported mono-functional Family V UDG homologs. Intriguingly, the enzyme can cleave DNA containing an AP site, thus suggesting that it might be involved in AP site repair. Biochemical data demonstrate that Sis-UDGV displays maximum activity for uracil removal at 45 °C ∼ 65 oC and at pH 8.0 ∼ 9.0. Furthermore, Sis-UDGV displays a substrate preference for uracil-containing ssDNA over uracil-containing dsDNA, but has no activity and weak activity for excising hypoxanthine from ssDNA and dsDNA, respectively. Importantly, we dissected the roles of seven conserved residues in Sis-UDGV by mutational analyses, demonstrating that residues D91, E117, E128, H167 and R192 are essential for catalysis. To our knowledge, it is the first report on the novel Family V UDG from Archaea with bi-functionality that harbors glycosylase/AP lyase activity.

A novel of new class II bacteriocin from Bacillus velezensis HN-Q-8 and its antibacterial activity on Streptomyces scabies.

Potato common scab is a main soil-borne disease of potato that can significantly reduce its quality. At present, it is still a challenge to control potato common scab in the field. To address this problem, the 972 family lactococcin (Lcn972) was screened from Bacillus velezensis HN-Q-8 in this study, and an Escherichia coli overexpression system was used to obtain Lcn972, which showed a significant inhibitory effect on Streptomyces scabies, with a minimum inhibitory concentration of 10.58 µg/mL. The stability test showed that Lcn972 is stable against UV radiation and high temperature. In addition, long-term storage at room temperature and 4°C had limited effects on its activity level. The antibacterial activity of Lcn972 was enhanced by Cu2+ and Ca2+, but decreased by protease K. The protein was completely inactivated by Fe2+. Cell membrane staining showed that Lcn972 damaged the cell membrane integrity of S. scabies. Scanning electron microscope (SEM) and transmission electron microscope (TEM) observations revealed that the hyphae of S. scabies treated with Lcn972 were deformed and adhered, the cell membrane was incomplete, the cytoplasm distribution was uneven, and the cell appeared hollow inside, which led to the death of S. scabies. In conclusion, we used bacteriocin for controlling potato common scab for the first time in this study, and it provides theoretical support for the further application of bacteriocin in the control of plant diseases.

Biochemical and Functional Characterization of an Iron-Containing Alcohol Dehydrogenase from Thermococcus barophilus Ch5.

Two iron-containing alcohol dehydrogenases (ADHs) are encoded in the genome of the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 (Tba ADH641 and Tba ADH547). In our previous publication, we reported biochemical characteristics and catalytic mechanism of Tba ADH547. Herein, we present evidence that Tba ADH641 possesses two activities for ethanol oxidization and acetaldehyde reduction at high temperature, capable of using NAD(H) and NADP(H) as coenzyme. Biochemical data show that Tba ADH641 possesses optimal reaction temperature, thermostability, divalent ion requirement, and substrate specificity distinct from Tba ADH547 and other iron-containing ADH homologues. However, Tba ADH641 and Tba ADH547 display same optimal reaction pH. Kinetic analyses demonstrate that Tba ADH641 displays higher catalytic efficiency for acetaldehyde reduction than that for ethanol oxidation, which is consistent with Tba ADH547. Mutational data demonstrate that residues D115, K118, E159, D190, and E215 in Tba ADH641, which has not been described to date, are necessary for enzyme activity, thus augmenting our understanding on catalytic mechanism of iron-containing ADH. Overall, our work demonstrates that Tba ADH641 is an iron-containing ADH with novel features, which is distinct from Tba ADH547, thus providing a potential biocatalyst for biotransformation reaction.

Serum IMA and LP-PLA2 Levels in Patients with Coronary Heart Disease and Their Correlation with the Degree of Myocardial Ischaemia and Their Diagnostic Value.

Purpose: To measure serum levels of ischaemia-modified albumin (IMA) and lipoprotein-associated phospholipase A2 (LP-PLA2) in patients with coronary heart disease (CHD) and to analyse their correlation with the degree of myocardial ischaemia and their diagnostic value. Methods: A sample of 150 patients diagnosed with CHD by coronary angiography in our hospital from March 2019 to September 2021 was taken as the CHD group. The patients were divided into acute myocardial infarction (AMI) group (n = 52), unstable angina pectoris (UAP) group (n = 54), and stable angina pectoris (SAP) group (n = 44) according to the degree of myocardial ischaemia, and then 50 healthy physical examination patients were selected as the health group during the same period. Serum C-reactive protein (CRP), interleukin-6 (IL-6), IMA, and LP-PLA2 levels were measured in each group separately. Multiple ordered logistic regression was used to analyse the factors influencing the degree of myocardial ischaemia in patients with CHD. Pearson correlation was used to analyse the correlation between serum IMA, LP-PLA2 levels and serum CRP, IL-6 levels in CHD patients. The diagnostic value of IMA alone, LP-PLA2 alone, and in combination for CHD was analysed using receiver operating characteristic (ROC) curves. Results: In terms of serum CRP, IL-6, IMA, and LP-PLA2 levels, the CHD group was higher than the health group, the AMI and UAP groups were higher than the SAP and health groups, and the AMI group was higher than the UAP group (P < 0.05). Multiple ordered logistic regression analysis showed that serum CRP, IL-6, IMA, and LP-PLA2 levels were all independent influences on the degree of myocardial ischaemia in patients with CHD (P < 0.05). Pearson correlation analysis showed a positive correlation between serum IMA, LP-PLA2 levels and serum CRP, IL-6 levels in CHD patients (P < 0.001). The area under curve (AUC) for serum IMA levels to predict myocardial ischaemia in patients with CHD was 0.754 (95% CI: 0.684-0.825), with a sensitivity of 61.3% and specificity of 84.0% when the best cut-off value was 0.453; the AUC for serum LP-PLA2 levels to predict myocardial ischaemia in patients with CHD was 0.747 (95% CI: 0.681-0.813), with a sensitivity of 62.0% and specificity of 82.0% when the optimal cut-off value was 0.440; and the AUC of IMA + LP-PLA2 for predicting myocardial ischaemia in patients with CHD was 0.892 (95% CI: 0.847-0.938), with a sensitivity of 86.7% and specificity of 80.0% when the optimal cut-off value was 0.667. The specificity was 80.0%. Conclusions: Serum IMA and LP-PLA2 levels are elevated in patients with CHD. Serum IMA and LP-PLA2 levels are closely related to the degree of myocardial ischaemia and its inflammatory level, and the combination of IMA + LP-PLA2 can improve the diagnosis efficacy of myocardial ischaemia in CHD patients.

A thermophilic 8-oxoguanine DNA glycosylase from Thermococcus barophilus Ch5 is a new member of AGOG DNA glycosylase family

8-Oxoguanine (8oxoG) in DNA is a major oxidized base that poses a severe threat to genome stability. To counteract the mutagenic effect generated by 8oxoG in DNA, cells have evolved 8oxoG DNA glycosylase (OGG) that can excise this oxidized base from DNA. Currently, OGG enzymes have been divided into three families: OGG1, OGG2 and AGOG (archaeal 8oxoG DNA glycosylase). Due to the limited reports, our understanding on AGOG enzymes remains incomplete. Herein, we present evidence that an AGOG from the hyperthermophilic euryarchaeon Ch5 (Tb-AGOG) excises 8oxoG from DNA at high temperature. The enzyme displays maximum efficiency at 75°C-95°C and at pH 9.0. As expected, Tb-AGOG is a bifunctional glycosylase that harbors glycosylase activity and AP (apurinic/apyrimidinic) lyase activity. Importantly, we reveal for the first time that residue D41 in Tb-AGOG is essential for 8oxoG excision and intermediate formation, but not essential for DNA binding or AP cleavage. Furthermore, residue E79 in Tb-AGOG is essential for 8oxoG excision and intermediate formation, and is partially involved in DNA binding and AP cleavage, which has not been described among the reported AGOG members to date. Overall, our work provides new insights into catalytic mechanism of AGOG enzymes.

Biochemical and functional characterization of an endonuclease III from Thermococcus barophilus Ch5.

Endonuclease III (EndoIII) is a bifunctional DNA glycosylase that is essential to excise thymine glycol (Tg) from DNA. Although EndoIII is widespread in bacteria, eukarya and Archaea, our understanding on archaeal EndoIII function remains relatively incomplete due to the limited reports. Herein, we characterized an EndoIII from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 (Tba-EndoIII) biochemically, demonstrating that the enzyme can excise Tg from dsDNA and display maximum activity at 50 ~ 70 °C and at pH 6.0 ~ 9.0 without the requirement of a divalent metal ion. Importantly, Tba-EndoIII differs from other reported archaeal EndoIII homologues in thermostability and salt requirement. As observed in other EndoIII homologues, the conserved residues D155 and H157 in Helix-hairpin-Helix motif of Tba-EndoIII are essential for Tg excision. Intriguingly, we first dissected that the conserved residues C215 and C221 in the Fe-S cluster loop in Tba-EndoIII are involved in intermediate formation and Tg excision. Additionally, we first revealed that the conserved residue L48 is flexible for intermediate formation and AP cleavage, but plays no detectable role in Tg excision. Overall, our work has revealed additional archaeal EndoIII function and catalytic mechanism.

Biochemical characterization and mutational analysis of a mismatch glycosylase from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5.

Mismatch glycosylase (MIG) can excise thymine and uracil from mutagenic T:G and U:G mispairs, which arise from cytosine and 5-methylcytosine deamination, respectively. Here, we present evidence that a thermostable MIG from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 (Tb-MIG) can remove thymine and uracil from T:G and U:G mispairs at high temperature, albeit at a low efficiency for U:G mispair. The enzyme displays maximum efficiency at 70 oC - 75 °C and pH 7.0-8.0. Tb-MIG is extremely thermostable, retaining 50% activity after heating at 100 oC for 2 hrs. In addition, Tb-MIG is a bifunctional glycosylase with an AP lyase activity, then resembles the MIG from the hyperthermopilic crenarchaeon Pyrobaculum aerophilium, but contrasts with the MIG from the hyperthermopilic crenarchaeon Aeropyrum pernix. Importantly, we show that residues Y133 and D151 in Tb-MIG are essential for thymine removal, and that residues A58, N153 and R156 are involved in thymine removal. Compared with the wild-type protein, the A58D and Y133K mutants display the increased AP lyase activity, confirming the essential roles played by the correspondingly conserved Asp and Lys in endonuclease III for AP site cleavage. Overall, our work is the first biochemical characterization of a hypthermophilic euryarchaeal MIG, augmenting our understanding on archaeal MIG function.

Biochemical and functional characterization of a thermostable RecJ exonuclease from Thermococcus gammatolerans.

RecJ is ubiquitous in bacteria and Archaea, and play an important role in DNA replication and repair. Currently, our understanding on biochemical function of archaeal RecJ is incomplete due to the limited reports. The genome of the hyperthermophilic and radioresistant euryarchaeon Thermococcus gammatolerans encodes one putative RecJ protein (Tga-RecJ). Herein, we report biochemical characteristics and catalytic mechanism of Tga-RecJ. Tga-RecJ can degrade ssDNA in the 5'-3' direction at high temperature as observed in Thermococcus kodakarensis RecJ and Pyrococcus furiosus RecJ, the two closest homologs of the enzyme. In contrasted to P. furiosus RecJ, Tga-RecJ lacks 3'-5' ssRNA exonuclease activity. Furthermore, maximum activity of Tga-RecJ is observed at 50 °C ~ 70 °C and pH 7.0-9.0 with Mn2+, and the enzyme is the most thermostable among the reported RecJ proteins. Additionally, the rates for hydrolyzing ssDNA by Tga-RecJ were estimated by kinetic analyses at 50 °C ~ 70 °C, thus revealing its activation energy (10.5 ± 0.6 kcal/mol), which is the first report on energy barrier for ssDNA degradation by RecJ. Mutational studies showed that the mutations of residues D36, D83, D105, H106, H107 and D166 in Tga-RecJ to alanine almost completely abolish its activity, thereby suggesting that these residues are essential for catalysis.

Biochemical characterization and mutational analysis of a novel flap endonuclease 1 from Thermococcus barophilus Ch5.

Flap endonuclease 1 (FEN1) plays important roles in DNA replication, repair and recombination. Herein, we report biochemical characteristics and catalytic mechanism of a novel FEN1 from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 (Tb-FEN1). As expected, the recombinant Tb-FEN1 can cleave 5'-flap DNA. However, the enzyme has no activity on cleaving pseudo Y DNA, which sharply contrasts with other archaeal and eukaryotic FEN1 homologs. Tb-FEN1 retains 24% relative activity after heating at 100 °C for 20 min, demonstrating that it is the most thermostable among all reported FEN1 proteins. The enzyme displays maximal activity in a wide range of pH from 7.0 to 9.5. The Tb-FEN1 activity is dependent on a divalent metal ion, among which Mg2+ and Mn2+ are optimal. Enzyme activity is inhibited by NaCl. Kinetic analyzes estimated that an activation energy for removal of 5'-flap from DNA by Tb-FEN1 was 35.7 ± 4.3 kcal/mol, which is the first report on energy barrier for excising 5'-flap from DNA by a FEN1 enzyme. Mutational studies demonstrate that the K87A, R94A and E154A amino acid substitutions abolish cleavage activity and reduce 5'-flap DNA binding efficiencies, suggesting that residues K87, R94, and E154 in Tb-FEN1 are essential for catalysis and DNA binding as well. Overall, Tb-FEN1 is an extremely thermostable endonuclease with unusual features.

Biochemical characterization and mutational studies of a thermostable endonuclease III from Sulfolobus islandicus REY15A.

Endonuclease III (EndoIII), which is ubiquitous in bacteria, Archaea and eukaryotes, plays an important role in excising thymine glycol (Tg) from DNA. Herein, we present evidence that an EndoIII from the hyperthermophilic crenarchaeon Sulfolobus islandicus REY15A (Sis-EndoIII) is capable of removing Tg from DNA at high temperature. Biochemical data show that the optimal temperature and pH of Sis-EndoIII are ca.70 °C and ca.7.0-8.0, respectively. Furthermore, the recombinant Sis-EndoIII retains relative weak activity without a divalent metal ion, and displays maximum activity in the presence of Mg2+ or Ca2+. Additionally, we first revealed the activation energy (Ea) of 39.7 ± 4.2 kcal/mol for Sis-EndoIII to remove Tg from dsDNA. As a bifunctional glycosylase, Sis-EndoIII possesses AP lyase activity in addition to glycosylase activity. Additionally, a covalent intermediate is formed between Sis-EndoIII and Tg-containing dsDNA. Mutational studies demonstrate that residues D50, K133 and D151 in Sis-EndoIII are responsible for removal of Tg from dsDNA and K133 and D151 are essential for formation of the covalent intermediate. To our knowledge, it is the first report of Tg excision by crenarchaeal EndoIII, thus augmenting our understanding on archaeal EndoIII function.

Repair of Hypoxanthine in DNA Revealed by DNA Glycosylases and Endonucleases From Hyperthermophilic Archaea.

Since hyperthermophilic Archaea (HA) thrive in high-temperature environments, which accelerate the rates of deamination of base in DNA, their genomic stability is facing a severe challenge. Hypoxanthine (Hx) is one of the common deaminated bases in DNA. Generally, replication of Hx in DNA before repaired causes AT → GC mutation. Biochemical data have demonstrated that 3-methyladenine DNA glycosylase II (AlkA) and Family V uracil DNA glycosylase (UDG) from HA could excise Hx from DNA, thus triggering a base excision repair (BER) process for Hx repair. Besides, three endonucleases have been reported from HA: Endonuclease V (EndoV), Endonuclease Q (EndoQ), and Endonuclease NucS (EndoNucS), capable of cleaving Hx-containing DNA, thereby providing alternative pathways for Hx repair. Both EndoV and EndoQ could cleave one DNA strand with Hx, thus forming a nick and further initiating an alternative excision repair (AER) process for the follow-up repair. By comparison, EndoNucS cleaves both strands of Hx-containing DNA in a restriction endonuclease manner, thus producing a double-stranded break (DSB). This created DSB might be repaired by homologous recombination (HR) or by a combination activity of DNA polymerase (DNA pol), flap endonuclease 1 (FEN1), and DNA ligase (DNA lig). Herein, we reviewed the most recent advances in repair of Hx in DNA triggered by DNA glycosylases and endonucleases from HA, and proposed future research directions.