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 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.

[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.

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.

Identification of a novel bifunctional uracil DNA glycosylase from Thermococcus barophilus Ch5.

Genomes of hyperthermophiles are facing a severe challenge due to increased deamination rates of cytosine induced by high temperature, which could be counteracted by base excision repair mediated by uracil DNA glycosylase (UDG) or other repair pathways. Our previous work has shown that the two UDGs (Tba UDG247 and Tba UDG194) encoded by the genome of the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 can remove uracil from DNA at high temperature. Herein, we provide evidence that Tba UDG247 is a novel bifunctional glycosylase which can excise uracil from DNA and further cleave the phosphodiester bo nd of the generated apurinic/apyrimidinic (AP) site, which has never been described to date. In addition to cleaving uracil-containing DNA, Tba UDG247 can also cleave AP-containing ssDNA although at lower efficiency, thereby suggesting that the enzyme might be involved in repair of AP site in DNA. Kinetic analyses showed that Tba UDG247 displays a faster rate for uracil excision than for AP cleavage, thus suggesting that cleaving AP site by the enzyme is a rate-limiting step for its bifunctionality. Phylogenetic analysis showed that Tba UDG247 is clustered on a separate branch distant from all the reported UDGs. Overall, we designated Tba UDG247 as the prototype of a novel family of bifunctional UDGs. KEY POINTS: We first reported a novel DNA glycosylase with bifunctionality. Tba UDG247 possesses an AP lyase activity.

Biochemical characterization and mutational studies of the 8-oxoguanine DNA glycosylase from the hyperthermophilic and radioresistant archaeon Thermococcus gammatolerans.

8-oxoguanine (GO) is a major lesion found in DNA that arises from guanine oxidation. The hyperthermophilic and radioresistant euryarchaeon Thermococcus gammatolerans encodes an archaeal GO DNA glycosylase (Tg-AGOG). Here, we characterized biochemically Tg-AGOG and probed its GO removal mechanism by mutational studies. Tg-AGOG can remove GO from DNA at high temperature through a ß-elimination reaction. The enzyme displays an optimal temperature, ca.85-95 °C, and an optimal pH, ca.7.0-8.5. In addition, Tg-AGOG activity is independent on a divalent metal ion. However, both Co2+ and Cu2+ inhibit its activity. The enzyme activity is also inhibited by NaCl. Furthermore, Tg-AGOG specifically cleaves GO-containing dsDNA in the order: GO:C, GO:T, GO:A, and GO:G. Moreover, the temperature dependence of cleavage rates of the enzyme was determined, and from this, the activation energy for GO removal from DNA was first estimated to be 16.9 ± 0.9 kcal/mol. In comparison with the wild-type Tg-AGOG, the R197A mutant has a reduced cleavage activity for GO-containing DNA, whereas both the P193A and F167A mutants exhibit similar cleavage activities for GO-containing DNA. While the mutations of P193 and F167 to Ala lead to increased binding, the mutation of R197 to Ala had no significant effect on binding. These observations suggest that residue R197 is involved in catalysis, and residues P193 and F167 are flexible for conformational change.

Biochemical characterization of a thermostable DNA ligase from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5.

DNA ligases are essential enzymes for DNA replication, repair, and recombination processes by catalyzing a nick-joining reaction in double-stranded DNA. The genome of the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 encodes a putative ATP-dependent DNA ligase (Tba ligase). Herein, we characterized the biochemical properties of the recombinant Tba ligase. The enzyme displays an optimal nick-joining activity at 65-70 °C and retains its DNA ligation activity even after heated at 100 °C for 2 h, suggesting the enzyme is a thermostable DNA ligase. The enzyme joins DNA over a wide pH spectrum ranging from 5.0-10.0, and its optimal pH is 6.0-9.0. Tba ligase activity is dependent on a divalent metal ion: Mn2+, Mg2+, or Ca2+ is an optimal ion for the enzyme activity. The enzyme activity is inhibited by NaCl with high concentrations. Tba ligase is ATP-dependent and can also use UTP as a weak cofactor; however, the enzyme with high concentrations could function without an additional nucleotide cofactor. Mass spectrometric result shows that the residue K250 of Tba ligase is AMPylated, suggesting that the enzyme is bound to AMP. The substitution of K250 of Tba ligase with Ala abolishes the enzyme activity. In addition, the mismatches at the first position 3' to the nick suppress Tba ligase activity more than those at the first position 5' to the nick. The enzyme also discriminates more effectively mismatches at 3' to the nick than those at 5' to the nick in a ligation cycling reaction, suggesting that the enzyme might have potential application in single nucleotide polymorphism.

The characterization and comparison of exopolysaccharides from two benthic diatoms with different biofilm formation abilities.

Exopolysaccharide (EPS) of two benthic diatoms, Amphora sp. and Stauroneis sp., with different biofilm formation abilities were investigated. The ratio of suspension-cells/biofilm-cells was employed to indicate the diatom biofilm formation abilities. The soluble EPS from the supernatant of whole culture, tightly bound EPS from floating cells, loosely and tightly bound EPS from biofilm cells were fractionated as SL-EPS, F-TB-EPS, BF-LB-EPS and BF-TB-EPS, respectively. The analysis for productions and monosaccharide compositions indicated that EPS from two diatoms were different in terms of the productions, distributions, and monomer compositions. Amphora sp. produced more (1.5-fold) total exopolysaccharides, but less (<0.4-fold) BF-TB-EPS than Stauroneis sp. The monosaccharides of the EPS from Amphora sp. were more diverse than those of Stauroneis sp., with 13 and 10 monomers, respectively. Neutral sugars, Glc, Xyl and Man, were abundant in Stauroneis sp., while Gal, Glc and Xyl were rich in Amphora sp. Uronic acid and hexosamine were present in all fractions of two diatoms, especially Glc-A being the most abundant monomer in SL-EPS of Amphora sp. It was proposed that the high content of uronic acid (especially Glc-A) might be crucial for the strong biofilm formation abilities of Amphora sp.

New Insights into DNA Polymerase Function Revealed by Phosphonoacetic Acid-Sensitive T4 DNA Polymerases.

The bacteriophage T4 DNA polymerase (pol) and the closely related RB69 DNA pol have been developed into model enzymes to study family B DNA pols. While all family B DNA pols have similar structures and share conserved protein motifs, the molecular mechanism underlying natural drug resistance of nonherpes family B DNA pols and drug sensitivity of herpes DNA pols remains unknown. In the present study, we constructed T4 phages containing G466S, Y460F, G466S/Y460F, P469S, and V475W mutations in DNA pol. These amino acid substitutions replace the residues in drug-resistant T4 DNA pol with residues found in drug-sensitive herpes family DNA pols. We investigated whether the T4 phages expressing the engineered mutant DNA pols were sensitive to the antiviral drug phosphonoacetic acid (PAA) and characterized the in vivo replication fidelity of the phage DNA pols. We found that G466S substitution marginally increased PAA sensitivity, whereas Y460F substitution conferred resistance. The phage expressing a double mutant G466S/Y460F DNA pol was more PAA-sensitive. V475W T4 DNA pol was highly sensitive to PAA, as was the case with V478W RB69 DNA pol. However, DNA replication was severely compromised, which resulted in the selection of phages expressing more robust DNA pols that have strong ability to replicate DNA and contain additional amino acid substitutions that suppress PAA sensitivity. Reduced replication fidelity was observed in all mutant phages expressing PAA-sensitive DNA pols. These observations indicate that PAA sensitivity and fidelity are balanced in DNA pols that can replicate DNA in different environments.

Biochemical characterization of a thermostable HNH endonuclease from deep-sea thermophilic bacteriophage GVE2.

His-Asn-His (HNH) proteins are a very common family of small nucleic acid-binding proteins that are generally associated with endonuclease activity and are found in all kingdoms of life. Although HNH endonucleases from mesophiles have been widely investigated, the biochemical functions of HNH endonucleases from thermophilic bacteriophages remain unknown. Here, we characterized the biochemical properties of a thermostable HNH endonuclease from deep-sea thermophilic bacteriophage GVE2. The recombinant GVE2 HNH endonuclease exhibited non-specific cleavage activity at high temperature. The optimal temperature of the GVE2 HNH endonuclease for cleaving DNA was 60-65 °C, and the enzyme retained its DNA cleavage activity even after heating at 100 °C for 30 min, suggesting the enzyme is a thermostable endonuclease. The GVE2 HNH endonuclease cleaved DNA over a wide pH spectrum, ranging from 5.5 to 9.0, and the optimal pH for the enzyme activity was 8.0-9.0. Furthermore, the GVE2 HNH endonuclease activity was dependent on a divalent metal ion. While the enzyme is inactive in the presence of Cu(2+), the GVE2 HNH endonuclease displayed cleavage activity of varied efficiency with Mn(2+), Mg(2+), Ca(2+), Fe(2+), Co(2+), Zn(2+), and Ni(2+). The GVE2 HNH endonuclease activity was inhibited by NaCl. This study provides the basis for determining the role of this endonuclease in life cycle of the bacteriophage GVE2 and suggests the potential application of the enzyme in molecular biology and biotechnology.

Fungal communities from the calcareous deep-sea sediments in the Southwest India Ridge revealed by Illumina sequencing technology.

The diversity and ecological significance of bacteria and archaea in deep-sea environments have been thoroughly investigated, but eukaryotic microorganisms in these areas, such as fungi, are poorly understood. To elucidate fungal diversity in calcareous deep-sea sediments in the Southwest India Ridge (SWIR), the internal transcribed spacer (ITS) regions of rRNA genes from two sediment metagenomic DNA samples were amplified and sequenced using the Illumina sequencing platform. The results revealed that 58-63 % and 36-42 % of the ITS sequences (97 % similarity) belonged to Basidiomycota and Ascomycota, respectively. These findings suggest that Basidiomycota and Ascomycota are the predominant fungal phyla in the two samples. We also found that Agaricomycetes, Leotiomycetes, and Pezizomycetes were the major fungal classes in the two samples. At the species level, Thelephoraceae sp. and Phialocephala fortinii were major fungal species in the two samples. Despite the low relative abundance, unidentified fungal sequences were also observed in the two samples. Furthermore, we found that there were slight differences in fungal diversity between the two sediment samples, although both were collected from the SWIR. Thus, our results demonstrate that calcareous deep-sea sediments in the SWIR harbor diverse fungi, which augment the fungal groups in deep-sea sediments. This is the first report of fungal communities in calcareous deep-sea sediments in the SWIR revealed by Illumina sequencing.

Archaeal DNA polymerases in biotechnology.

DNA polymerase (pol) is a ubiquitous enzyme that synthesizes DNA strands in all living cells. In vitro, DNA pol is used for DNA manipulation, including cloning, PCR, site-directed mutagenesis, sequencing, and several other applications. Family B archaeal DNA pols have been widely used for molecular biological methods. Biochemical and structural studies reveal that each archaeal DNA pol has different characteristics with respect to fidelity, processivity and thermostability. Due to their high fidelity and strong thermostability, family B archaeal DNA pols have the extensive application on high-fidelity PCR, DNA sequencing, and site-directed mutagenesis while family Y archaeal DNA pols have the potential for error-prone PCR and random mutagenesis because of their low fidelity and strong thermostability. This information combined with mutational analysis has been used to construct novel DNA pols with altered properties that enhance their use as biotechnological reagents. In this review, we focus on the development and use of family B archaeal DNA pols.

Sulfolobus replication factor C stimulates the activity of DNA polymerase B1.

Replication factor C (RFC) is known to function in loading proliferating cell nuclear antigen (PCNA) onto primed DNA, allowing PCNA to tether DNA polymerase for highly processive DNA synthesis in eukaryotic and archaeal replication. In this report, we show that an RFC complex from the hyperthermophilic archaea of the genus Sulfolobus physically interacts with DNA polymerase B1 (PolB1) and enhances both the polymerase and 3'-5' exonuclease activities of PolB1 in an ATP-independent manner. Stimulation of the PolB1 activity by RFC is independent of the ability of RFC to bind DNA but is consistent with the ability of RFC to facilitate DNA binding by PolB1 through protein-protein interaction. These results suggest that Sulfolobus RFC may play a role in recruiting DNA polymerase for efficient primer extension, in addition to clamp loading, during DNA replication.

A novel Mre11 protein from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 possesses 5'-3' exonuclease and endonuclease activities.

Mre11 is one of important proteins that are involved in DNA repair and recombination by processing DNA ends to produce 3'-single stranded DNA, thus providing a platform for other DNA repair and recombination proteins. In this work, we characterized the Mre11 protein from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 (Tba-Mre11) biochemically and dissected the roles of its four conserved residues, which is the first report on Mre11 proteins from Thermococcus. Tba-Mre11 possesses exonuclease activity for degrading ssDNA and dsDNA in the 5'-3' direction, which contrasts with other reported Mre11 homologs. Maximum degradation efficiency was observed with Mn2+ at 80 °C and at pH 7.5-9.5. In addition to possessing 5'-3' exonuclease activity, Tba-Mre11 has endonuclease activity that nicks plasmid DNA and circular ssDNA. Mutational data show that residues D10, D51 and N86 in Tba-Mre11 are essential for DNA degradation since almost no activity was observed for the D10A, D51A and N86A mutants. By comparison, residue D44 in Tba-Mre11 is not responsible for DNA degradation since the D44A mutant possessed the similar WT protein activity. Notably, the D44A mutant almost completely abolished the ability to bind DNA, suggesting that residue D44 is essential for binding DNA.

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.

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.

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.

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.

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.