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GenScript corporation thermococcus sp. 9°n rnaseh2 gene

Archaea require an <t>RNaseH2-initiated</t> RER pathway. A , genomic DNA from wild-type and ΔRNaseH2 T. kodakarensis ( Tko ) cells was treated with either 0.3 m NaCl or 0.3 m NaOH, separated on a 1% alkaline-agarose gel, and visualized using SYBR Gold staining. The fluorescence intensity distribution was quantified using ImageQuant software. B, primed M13mp18 ssDNA was fully extended by either Thermococcus sp. <t>9°N</t> PolB or PolD with dNTPs or dNTPs/excess rNTPs (see “Experimental procedures”) with [ 32 P]dCTP in place of dCTP for phosphorimaging. Full extension products were visualized by neutral agarose and rNMP incorporation was assessed by 0.3 m NaOH treatment and alkaline-agarose electrophoresis. C, RNaseH2 activity in Tko extracts was monitored by CE using a 50-bp dsDNA substrate with an embedded rGMP nucleotide, a 5′-FAM label, and a 3′-MAX label. Reactions were carried out over a time course from 15 s to 20 min at 60 °C. A subset of representative CE traces are shown indicating the formation of 21- and 29-nt RNaseH2 products. 3′-MAX-labeled Fen1 flap cleavage products (<29 nt) are also observed. D, the formation of the 29-nt MAX product was quantified over time for both wild-type ( red ) and ΔRNaseH2 ( blue ) extracts. Data are the average of three biological replicates and error bars indicate standard deviation.

Thermococcus Sp. 9°N Rnaseh2 Gene, supplied by GenScript corporation, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Defining the RNaseH2 enzyme-initiated ribonucleotide excision repair pathway in Archaea"

Article Title: Defining the RNaseH2 enzyme-initiated ribonucleotide excision repair pathway in Archaea

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M117.783472

Archaea require an RNaseH2-initiated RER pathway. A , genomic DNA from wild-type and ΔRNaseH2 T. kodakarensis ( Tko ) cells was treated with either 0.3 m NaCl or 0.3 m NaOH, separated on a 1% alkaline-agarose gel, and visualized using SYBR Gold staining. The fluorescence intensity distribution was quantified using ImageQuant software. B, primed M13mp18 ssDNA was fully extended by either Thermococcus sp. 9°N PolB or PolD with dNTPs or dNTPs/excess rNTPs (see “Experimental procedures”) with [ 32 P]dCTP in place of dCTP for phosphorimaging. Full extension products were visualized by neutral agarose and rNMP incorporation was assessed by 0.3 m NaOH treatment and alkaline-agarose electrophoresis. C, RNaseH2 activity in Tko extracts was monitored by CE using a 50-bp dsDNA substrate with an embedded rGMP nucleotide, a 5′-FAM label, and a 3′-MAX label. Reactions were carried out over a time course from 15 s to 20 min at 60 °C. A subset of representative CE traces are shown indicating the formation of 21- and 29-nt RNaseH2 products. 3′-MAX-labeled Fen1 flap cleavage products (<29 nt) are also observed. D, the formation of the 29-nt MAX product was quantified over time for both wild-type ( red ) and ΔRNaseH2 ( blue ) extracts. Data are the average of three biological replicates and error bars indicate standard deviation.

Figure Legend Snippet: Archaea require an RNaseH2-initiated RER pathway. A , genomic DNA from wild-type and ΔRNaseH2 T. kodakarensis ( Tko ) cells was treated with either 0.3 m NaCl or 0.3 m NaOH, separated on a 1% alkaline-agarose gel, and visualized using SYBR Gold staining. The fluorescence intensity distribution was quantified using ImageQuant software. B, primed M13mp18 ssDNA was fully extended by either Thermococcus sp. 9°N PolB or PolD with dNTPs or dNTPs/excess rNTPs (see “Experimental procedures”) with [ 32 P]dCTP in place of dCTP for phosphorimaging. Full extension products were visualized by neutral agarose and rNMP incorporation was assessed by 0.3 m NaOH treatment and alkaline-agarose electrophoresis. C, RNaseH2 activity in Tko extracts was monitored by CE using a 50-bp dsDNA substrate with an embedded rGMP nucleotide, a 5′-FAM label, and a 3′-MAX label. Reactions were carried out over a time course from 15 s to 20 min at 60 °C. A subset of representative CE traces are shown indicating the formation of 21- and 29-nt RNaseH2 products. 3′-MAX-labeled Fen1 flap cleavage products (<29 nt) are also observed. D, the formation of the 29-nt MAX product was quantified over time for both wild-type ( red ) and ΔRNaseH2 ( blue ) extracts. Data are the average of three biological replicates and error bars indicate standard deviation.

Techniques Used: Agarose Gel Electrophoresis, Staining, Fluorescence, Software, Electrophoresis, Activity Assay, Labeling, Standard Deviation

RNaseH2 cleavage is not the rate-limiting step in RER. A , for steady-state kinetics, a 5-fold excess of 50 bp, 5′-FAM, 3′-MAX-labeled rGMP RER substrate was rapidly mixed with purified Thermococcus sp. 9°N RNaseH2 in an RQF instrument at 60 °C. The reaction was quenched with 50 m m EDTA. B, the conversion of the 50-nt, dual-labeled rG substrate to 5′-FAM and 3′-MAX cleavage products was monitored over time using CE. A subset of representative traces from a time course are shown. C, the yield of 5′-FAM product was graphed as a function of time and fit to to obtain k ss = 0.06 s −1 and the active enzyme concentration ( A ) = 5.5 n m . A representative plot for the rG substrate is shown and replicates for rG and the other ribonucleotide substrates are shown in supplemental Fig. S1 . D, pre-steady state kinetics were performed with purified Thermococcus sp. 9°N RNaseH2 chelated for metal ion, pre-bound to the 50-bp RER substrates with different embedded rNMPs. The enzyme was in 3-fold excess to the DNA substrate. The pre-bound DNA-RNaseH2 complex was rapidly mixed with buffer containing MgCl 2 in an RQF at 60 °C and quenched with 50 m m EDTA. Cleavage products were visualized by CE. E, the yield of 5′-FAM product was graphed as a function of time and fit to to obtain k cleavage for rG, rA, rC, and rU (222, 196, 447, and 426 s −1 ). A representative plot for the rG substrate is shown. Technical replicates of the pre-steady-state experiments for the rG and other ribonucleotide substrates are shown in supplemental Fig. S2 .

Figure Legend Snippet: RNaseH2 cleavage is not the rate-limiting step in RER. A , for steady-state kinetics, a 5-fold excess of 50 bp, 5′-FAM, 3′-MAX-labeled rGMP RER substrate was rapidly mixed with purified Thermococcus sp. 9°N RNaseH2 in an RQF instrument at 60 °C. The reaction was quenched with 50 m m EDTA. B, the conversion of the 50-nt, dual-labeled rG substrate to 5′-FAM and 3′-MAX cleavage products was monitored over time using CE. A subset of representative traces from a time course are shown. C, the yield of 5′-FAM product was graphed as a function of time and fit to to obtain k ss = 0.06 s −1 and the active enzyme concentration ( A ) = 5.5 n m . A representative plot for the rG substrate is shown and replicates for rG and the other ribonucleotide substrates are shown in supplemental Fig. S1 . D, pre-steady state kinetics were performed with purified Thermococcus sp. 9°N RNaseH2 chelated for metal ion, pre-bound to the 50-bp RER substrates with different embedded rNMPs. The enzyme was in 3-fold excess to the DNA substrate. The pre-bound DNA-RNaseH2 complex was rapidly mixed with buffer containing MgCl 2 in an RQF at 60 °C and quenched with 50 m m EDTA. Cleavage products were visualized by CE. E, the yield of 5′-FAM product was graphed as a function of time and fit to to obtain k cleavage for rG, rA, rC, and rU (222, 196, 447, and 426 s −1 ). A representative plot for the rG substrate is shown. Technical replicates of the pre-steady-state experiments for the rG and other ribonucleotide substrates are shown in supplemental Fig. S2 .

Techniques Used: Labeling, Purification, Concentration Assay

Figure Legend Snippet: 9°N RNaseH2 kinetic parameters

Techniques Used:

Dual-label fluorescence assay to monitor post-RNaseH2 RER by capillary electrophoresis. A, the RER substrate was generated by annealing a 44-nt (5′-MAX labeled) oligonucleotide and a 90-nt (3′-FAM labeled with a 5′-phosphate-rGMP) oligonucleotide to ssM13 DNA. Annealed oligos form a DNA substrate containing a nick with 3′-OH and 5′-phosphate-rG termini to mimic DNA nicked by RNaseH2. On CE, the double-stranded DNA is denatured and ssDNA oligonucleotides can be visualized individually. On the right is a hypothetical CE trace representing the expected result with two individual MAX and FAM oligonucleotide peaks. B, when a strand-displacing DNA polymerase is added, 5′-MAX-labeled strand displacement products larger than 44-nt can be observed. C, when a flap endonuclease is added, 3′-FAM products smaller than 90-nt (predominantly by 1–2 nt) are observed. D, full sealing and repair by ligation results in a FAM- and MAX-labeled 134-nt product.

Figure Legend Snippet: Dual-label fluorescence assay to monitor post-RNaseH2 RER by capillary electrophoresis. A, the RER substrate was generated by annealing a 44-nt (5′-MAX labeled) oligonucleotide and a 90-nt (3′-FAM labeled with a 5′-phosphate-rGMP) oligonucleotide to ssM13 DNA. Annealed oligos form a DNA substrate containing a nick with 3′-OH and 5′-phosphate-rG termini to mimic DNA nicked by RNaseH2. On CE, the double-stranded DNA is denatured and ssDNA oligonucleotides can be visualized individually. On the right is a hypothetical CE trace representing the expected result with two individual MAX and FAM oligonucleotide peaks. B, when a strand-displacing DNA polymerase is added, 5′-MAX-labeled strand displacement products larger than 44-nt can be observed. C, when a flap endonuclease is added, 3′-FAM products smaller than 90-nt (predominantly by 1–2 nt) are observed. D, full sealing and repair by ligation results in a FAM- and MAX-labeled 134-nt product.

Techniques Used: Fluorescence, Electrophoresis, Generated, Labeling, Ligation

Archaeal RER reconstituted in vitro . A, reaction schematic. The substrate depicted in is incubated with purified Thermococcus sp. 9°N proteins including PCNA, RFC, and different combinations of PolB, PolD, Fen1, and DNA ligase. Reactions were incubated over a time course from 0 to 30 min at 60 °C and repair was monitored by appearance of the 134-nt FAM/MAX-labeled DNA product by CE. B, representative CE traces for each reaction condition at the 30-min time point are shown. C, quantification of the conversion of 90-nt FAM substrate to 134-nt FAM/MAX product at the 30-min time point. Data are the average of three independent experiments with S.D.

Figure Legend Snippet: Archaeal RER reconstituted in vitro . A, reaction schematic. The substrate depicted in is incubated with purified Thermococcus sp. 9°N proteins including PCNA, RFC, and different combinations of PolB, PolD, Fen1, and DNA ligase. Reactions were incubated over a time course from 0 to 30 min at 60 °C and repair was monitored by appearance of the 134-nt FAM/MAX-labeled DNA product by CE. B, representative CE traces for each reaction condition at the 30-min time point are shown. C, quantification of the conversion of 90-nt FAM substrate to 134-nt FAM/MAX product at the 30-min time point. Data are the average of three independent experiments with S.D.

Techniques Used: In Vitro, Incubation, Purification, Labeling

Simplified models of ribonucleotide excision repair in Eukarya ( A ), Archaea ( B ), and Bacteria ( C ). A, rNMPs are incorporated into eukaryotic genomic DNA by any of the replicative polymerases (Polϵ, Polδ, or Polα) with the incorporation frequencies shown ( , , ). The RER pathway begins with incision by the heterotrimeric RNaseH2 followed by strand displacement synthesis by either of the replicative polymerases Polϵ or Polδ, flap cleavage by Fen1 or Exo1, and sealing by DNA ligase I. B, archaeal genomic DNA acquires rNMPs primarily through incorporation by the leading and lagging strand polymerase PolD at a rate of 1 rN in 1,500 nucleotides synthesized. Monomeric RNaseH2 nicks DNA at rNMP sites. Following cleavage, strand displacement synthesis by PolB creates a flap that is cleaved by Fen1 and DNA ligase seals the resulting nick. C, in bacteria, the replicative polymerase PolIII incorporates rNMPs in genomic DNA and monomeric RNaseHII nicks at these sites. PolI then fulfills two functions in RER by performing both strand displacement synthesis and flap cleavage. DNA ligase then seals the nick.

Figure Legend Snippet: Simplified models of ribonucleotide excision repair in Eukarya ( A ), Archaea ( B ), and Bacteria ( C ). A, rNMPs are incorporated into eukaryotic genomic DNA by any of the replicative polymerases (Polϵ, Polδ, or Polα) with the incorporation frequencies shown ( , , ). The RER pathway begins with incision by the heterotrimeric RNaseH2 followed by strand displacement synthesis by either of the replicative polymerases Polϵ or Polδ, flap cleavage by Fen1 or Exo1, and sealing by DNA ligase I. B, archaeal genomic DNA acquires rNMPs primarily through incorporation by the leading and lagging strand polymerase PolD at a rate of 1 rN in 1,500 nucleotides synthesized. Monomeric RNaseH2 nicks DNA at rNMP sites. Following cleavage, strand displacement synthesis by PolB creates a flap that is cleaved by Fen1 and DNA ligase seals the resulting nick. C, in bacteria, the replicative polymerase PolIII incorporates rNMPs in genomic DNA and monomeric RNaseHII nicks at these sites. PolI then fulfills two functions in RER by performing both strand displacement synthesis and flap cleavage. DNA ligase then seals the nick.

Techniques Used: Bacteria, Synthesized

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Journal: The Journal of Biological Chemistry

Article Title: Defining the RNaseH2 enzyme-initiated ribonucleotide excision repair pathway in Archaea

doi: 10.1074/jbc.M117.783472

Figure Lengend Snippet: Archaea require an RNaseH2-initiated RER pathway. A , genomic DNA from wild-type and ΔRNaseH2 T. kodakarensis ( Tko ) cells was treated with either 0.3 m NaCl or 0.3 m NaOH, separated on a 1% alkaline-agarose gel, and visualized using SYBR Gold staining. The fluorescence intensity distribution was quantified using ImageQuant software. B, primed M13mp18 ssDNA was fully extended by either Thermococcus sp. 9°N PolB or PolD with dNTPs or dNTPs/excess rNTPs (see “Experimental procedures”) with [ 32 P]dCTP in place of dCTP for phosphorimaging. Full extension products were visualized by neutral agarose and rNMP incorporation was assessed by 0.3 m NaOH treatment and alkaline-agarose electrophoresis. C, RNaseH2 activity in Tko extracts was monitored by CE using a 50-bp dsDNA substrate with an embedded rGMP nucleotide, a 5′-FAM label, and a 3′-MAX label. Reactions were carried out over a time course from 15 s to 20 min at 60 °C. A subset of representative CE traces are shown indicating the formation of 21- and 29-nt RNaseH2 products. 3′-MAX-labeled Fen1 flap cleavage products (<29 nt) are also observed. D, the formation of the 29-nt MAX product was quantified over time for both wild-type ( red ) and ΔRNaseH2 ( blue ) extracts. Data are the average of three biological replicates and error bars indicate standard deviation.

Article Snippet: The Thermococcus sp. 9°N RNaseH2 gene was codon optimized for expression in E. coli , constructed synthetically (Genscript, Piscataway, NJ), and cloned into pAII17 plasmid vector ( ) cleaved with NdeI and BamHI to produce plasmid pESY.

Techniques: Agarose Gel Electrophoresis, Staining, Fluorescence, Software, Electrophoresis, Activity Assay, Labeling, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Defining the RNaseH2 enzyme-initiated ribonucleotide excision repair pathway in Archaea

doi: 10.1074/jbc.M117.783472

Figure Lengend Snippet: RNaseH2 cleavage is not the rate-limiting step in RER. A , for steady-state kinetics, a 5-fold excess of 50 bp, 5′-FAM, 3′-MAX-labeled rGMP RER substrate was rapidly mixed with purified Thermococcus sp. 9°N RNaseH2 in an RQF instrument at 60 °C. The reaction was quenched with 50 m m EDTA. B, the conversion of the 50-nt, dual-labeled rG substrate to 5′-FAM and 3′-MAX cleavage products was monitored over time using CE. A subset of representative traces from a time course are shown. C, the yield of 5′-FAM product was graphed as a function of time and fit to to obtain k ss = 0.06 s −1 and the active enzyme concentration ( A ) = 5.5 n m . A representative plot for the rG substrate is shown and replicates for rG and the other ribonucleotide substrates are shown in supplemental Fig. S1 . D, pre-steady state kinetics were performed with purified Thermococcus sp. 9°N RNaseH2 chelated for metal ion, pre-bound to the 50-bp RER substrates with different embedded rNMPs. The enzyme was in 3-fold excess to the DNA substrate. The pre-bound DNA-RNaseH2 complex was rapidly mixed with buffer containing MgCl 2 in an RQF at 60 °C and quenched with 50 m m EDTA. Cleavage products were visualized by CE. E, the yield of 5′-FAM product was graphed as a function of time and fit to to obtain k cleavage for rG, rA, rC, and rU (222, 196, 447, and 426 s −1 ). A representative plot for the rG substrate is shown. Technical replicates of the pre-steady-state experiments for the rG and other ribonucleotide substrates are shown in supplemental Fig. S2 .

Techniques: Labeling, Purification, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Defining the RNaseH2 enzyme-initiated ribonucleotide excision repair pathway in Archaea

doi: 10.1074/jbc.M117.783472

Figure Lengend Snippet: 9°N RNaseH2 kinetic parameters

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Defining the RNaseH2 enzyme-initiated ribonucleotide excision repair pathway in Archaea

doi: 10.1074/jbc.M117.783472

Figure Lengend Snippet: Dual-label fluorescence assay to monitor post-RNaseH2 RER by capillary electrophoresis. A, the RER substrate was generated by annealing a 44-nt (5′-MAX labeled) oligonucleotide and a 90-nt (3′-FAM labeled with a 5′-phosphate-rGMP) oligonucleotide to ssM13 DNA. Annealed oligos form a DNA substrate containing a nick with 3′-OH and 5′-phosphate-rG termini to mimic DNA nicked by RNaseH2. On CE, the double-stranded DNA is denatured and ssDNA oligonucleotides can be visualized individually. On the right is a hypothetical CE trace representing the expected result with two individual MAX and FAM oligonucleotide peaks. B, when a strand-displacing DNA polymerase is added, 5′-MAX-labeled strand displacement products larger than 44-nt can be observed. C, when a flap endonuclease is added, 3′-FAM products smaller than 90-nt (predominantly by 1–2 nt) are observed. D, full sealing and repair by ligation results in a FAM- and MAX-labeled 134-nt product.

Techniques: Fluorescence, Electrophoresis, Generated, Labeling, Ligation

Journal: The Journal of Biological Chemistry

Article Title: Defining the RNaseH2 enzyme-initiated ribonucleotide excision repair pathway in Archaea

doi: 10.1074/jbc.M117.783472

Figure Lengend Snippet: Archaeal RER reconstituted in vitro . A, reaction schematic. The substrate depicted in is incubated with purified Thermococcus sp. 9°N proteins including PCNA, RFC, and different combinations of PolB, PolD, Fen1, and DNA ligase. Reactions were incubated over a time course from 0 to 30 min at 60 °C and repair was monitored by appearance of the 134-nt FAM/MAX-labeled DNA product by CE. B, representative CE traces for each reaction condition at the 30-min time point are shown. C, quantification of the conversion of 90-nt FAM substrate to 134-nt FAM/MAX product at the 30-min time point. Data are the average of three independent experiments with S.D.

Techniques: In Vitro, Incubation, Purification, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Defining the RNaseH2 enzyme-initiated ribonucleotide excision repair pathway in Archaea

doi: 10.1074/jbc.M117.783472

Figure Lengend Snippet: Simplified models of ribonucleotide excision repair in Eukarya ( A ), Archaea ( B ), and Bacteria ( C ). A, rNMPs are incorporated into eukaryotic genomic DNA by any of the replicative polymerases (Polϵ, Polδ, or Polα) with the incorporation frequencies shown ( , , ). The RER pathway begins with incision by the heterotrimeric RNaseH2 followed by strand displacement synthesis by either of the replicative polymerases Polϵ or Polδ, flap cleavage by Fen1 or Exo1, and sealing by DNA ligase I. B, archaeal genomic DNA acquires rNMPs primarily through incorporation by the leading and lagging strand polymerase PolD at a rate of 1 rN in 1,500 nucleotides synthesized. Monomeric RNaseH2 nicks DNA at rNMP sites. Following cleavage, strand displacement synthesis by PolB creates a flap that is cleaved by Fen1 and DNA ligase seals the resulting nick. C, in bacteria, the replicative polymerase PolIII incorporates rNMPs in genomic DNA and monomeric RNaseHII nicks at these sites. PolI then fulfills two functions in RER by performing both strand displacement synthesis and flap cleavage. DNA ligase then seals the nick.

Techniques: Bacteria, Synthesized

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