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    Structured Review

    New England Biolabs atpase assays hydrolysis
    Mth810 is the archaeal orthologue of metazoan Hel308 in sequence and minimal helicase function. ( A ) Cartoon showing common features of Hel308 from archaea (Hel308a), human (hHel308) and the N-terminal domain of human PolQ. Helicase motifs, including the Q-motif ( 53 ), are labelled and the Hel308a sequences are given for motif I and IVa with mutagenized residues in bold and underlined. ( B ) Sequence details in helicase motifs V and VI that confirm Hel308a as a Hel308/Mus308 family rather than a RecQ helicase. The corresponding motif of human BLM helicase is shown for comparison (hBLM). In each motif peculiar residues conserved in Hel308/Mus308 helicases are in bold. Motif IVa is highly conserved in RecQ and Hel308 proteins. Invariant residues are in bold and highly conserved residues are underlined. Aligned with Hel308a, human Hel308 and human PolQ are Hel308 from Caenorhabditis elegans (CeHel308), E.coli RecQ (EcRecQ) and a human RecQ, BLM (HsBLM). ( C ) SDS–PAGE gel (10% acrylamide) showing purified recombinant Hel308a (arrowed) from Methanothermobacter . Marker sizes are given on the left of the panel. ( D ) <t>ATPase</t> activity of Hel308a measured as a function of time in reactions containing no DNA (filled diamond), dsDNA (open square) or ssDNA (open circles). Error bars are derived from the means of three independent experiments. ( E ) Unwinding reactions of Hel308a on 3′-ssDNA-tailed duplex (i), 5′-ssDNA-tailed duplex (ii) and untailed duplex (iii). Reactions were for 20 min at 45°C containing 2 nM DNA, with 32 P-labelled strand indicated by filled circle, 5 mM MgCl 2 , 5 mM <t>ATP</t> and zero (lane a); 1, 5, 10, 25 and 50 nM Hel308a (lanes b–f).
    Atpase Assays Hydrolysis, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/atpase assays hydrolysis/product/New England Biolabs
    Average 85 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    atpase assays hydrolysis - by Bioz Stars, 2020-11
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    Images

    1) Product Images from "Archaeal Hel308 helicase targets replication forks in vivo and in vitro and unwinds lagging strands"

    Article Title: Archaeal Hel308 helicase targets replication forks in vivo and in vitro and unwinds lagging strands

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki685

    Mth810 is the archaeal orthologue of metazoan Hel308 in sequence and minimal helicase function. ( A ) Cartoon showing common features of Hel308 from archaea (Hel308a), human (hHel308) and the N-terminal domain of human PolQ. Helicase motifs, including the Q-motif ( 53 ), are labelled and the Hel308a sequences are given for motif I and IVa with mutagenized residues in bold and underlined. ( B ) Sequence details in helicase motifs V and VI that confirm Hel308a as a Hel308/Mus308 family rather than a RecQ helicase. The corresponding motif of human BLM helicase is shown for comparison (hBLM). In each motif peculiar residues conserved in Hel308/Mus308 helicases are in bold. Motif IVa is highly conserved in RecQ and Hel308 proteins. Invariant residues are in bold and highly conserved residues are underlined. Aligned with Hel308a, human Hel308 and human PolQ are Hel308 from Caenorhabditis elegans (CeHel308), E.coli RecQ (EcRecQ) and a human RecQ, BLM (HsBLM). ( C ) SDS–PAGE gel (10% acrylamide) showing purified recombinant Hel308a (arrowed) from Methanothermobacter . Marker sizes are given on the left of the panel. ( D ) ATPase activity of Hel308a measured as a function of time in reactions containing no DNA (filled diamond), dsDNA (open square) or ssDNA (open circles). Error bars are derived from the means of three independent experiments. ( E ) Unwinding reactions of Hel308a on 3′-ssDNA-tailed duplex (i), 5′-ssDNA-tailed duplex (ii) and untailed duplex (iii). Reactions were for 20 min at 45°C containing 2 nM DNA, with 32 P-labelled strand indicated by filled circle, 5 mM MgCl 2 , 5 mM ATP and zero (lane a); 1, 5, 10, 25 and 50 nM Hel308a (lanes b–f).
    Figure Legend Snippet: Mth810 is the archaeal orthologue of metazoan Hel308 in sequence and minimal helicase function. ( A ) Cartoon showing common features of Hel308 from archaea (Hel308a), human (hHel308) and the N-terminal domain of human PolQ. Helicase motifs, including the Q-motif ( 53 ), are labelled and the Hel308a sequences are given for motif I and IVa with mutagenized residues in bold and underlined. ( B ) Sequence details in helicase motifs V and VI that confirm Hel308a as a Hel308/Mus308 family rather than a RecQ helicase. The corresponding motif of human BLM helicase is shown for comparison (hBLM). In each motif peculiar residues conserved in Hel308/Mus308 helicases are in bold. Motif IVa is highly conserved in RecQ and Hel308 proteins. Invariant residues are in bold and highly conserved residues are underlined. Aligned with Hel308a, human Hel308 and human PolQ are Hel308 from Caenorhabditis elegans (CeHel308), E.coli RecQ (EcRecQ) and a human RecQ, BLM (HsBLM). ( C ) SDS–PAGE gel (10% acrylamide) showing purified recombinant Hel308a (arrowed) from Methanothermobacter . Marker sizes are given on the left of the panel. ( D ) ATPase activity of Hel308a measured as a function of time in reactions containing no DNA (filled diamond), dsDNA (open square) or ssDNA (open circles). Error bars are derived from the means of three independent experiments. ( E ) Unwinding reactions of Hel308a on 3′-ssDNA-tailed duplex (i), 5′-ssDNA-tailed duplex (ii) and untailed duplex (iii). Reactions were for 20 min at 45°C containing 2 nM DNA, with 32 P-labelled strand indicated by filled circle, 5 mM MgCl 2 , 5 mM ATP and zero (lane a); 1, 5, 10, 25 and 50 nM Hel308a (lanes b–f).

    Techniques Used: Sequencing, SDS Page, Purification, Recombinant, Marker, Activity Assay, Derivative Assay

    2) Product Images from "Archaeal Hel308 helicase targets replication forks in vivo and in vitro and unwinds lagging strands"

    Article Title: Archaeal Hel308 helicase targets replication forks in vivo and in vitro and unwinds lagging strands

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki685

    Hel308a targets DNA forks for unwinding and binds to duplex substrates with branchpoints. ( A ) Time-course unwinding of four substrates acted on by Hel308a: fork-2 (filled circles), fork-1 (filled squares), 3′-ssDNA-tailed duplex (open circles) and Holliday junction (open squares). Substrates used are annotated to the right of the graph. Reactions were at 45°C for the times shown and contained 2 nM DNA, 5 mM MgCl 2 , 5 mM ATP and 20 nM Hel308a. Error bars are mostly hidden by data points and were derived from means of three independent assays. ( B ) Gel-retardation binding assays of Hel308a on fully base-paired, static Holliday junction (lanes a–f), static Holliday junction containing a backbone nick indicated by an arrowhead (lanes g–l), fully base-paired fork-3 (lanes m–r), fork-3 lacking a leading strand (lanes s–x) and fork-3 lacking a lagging strand (lanes y–4). Reactions were at 45°C for 10 min in 1 mM magnesium and contained 2 nM DNA and Hel308a at 0, 1, 2, 10, 50 and100 nM. ( C ) Hel308a K51L protein is unable to unwind fork-2 (lanes g–l), compared with wild-type protein (lanes a–f). Reactions were for 20 min at 45°C containing 2 nM DNA, 5 mM MgCl 2 , 5 mM ATP and 0, 1, 5, 10, 25 or 50 nM Hel308a. Binding of wild-type Hel308a (lanes m–o) and K51L Hel308a (lanes p–r) to fork 2 were in reactions for 10 min at 45°C containing 5 mM MgCl 2 , 2 nM fork-2 (labelled on strand 1) and 0, 10 or 100 nM Hel308a protein.
    Figure Legend Snippet: Hel308a targets DNA forks for unwinding and binds to duplex substrates with branchpoints. ( A ) Time-course unwinding of four substrates acted on by Hel308a: fork-2 (filled circles), fork-1 (filled squares), 3′-ssDNA-tailed duplex (open circles) and Holliday junction (open squares). Substrates used are annotated to the right of the graph. Reactions were at 45°C for the times shown and contained 2 nM DNA, 5 mM MgCl 2 , 5 mM ATP and 20 nM Hel308a. Error bars are mostly hidden by data points and were derived from means of three independent assays. ( B ) Gel-retardation binding assays of Hel308a on fully base-paired, static Holliday junction (lanes a–f), static Holliday junction containing a backbone nick indicated by an arrowhead (lanes g–l), fully base-paired fork-3 (lanes m–r), fork-3 lacking a leading strand (lanes s–x) and fork-3 lacking a lagging strand (lanes y–4). Reactions were at 45°C for 10 min in 1 mM magnesium and contained 2 nM DNA and Hel308a at 0, 1, 2, 10, 50 and100 nM. ( C ) Hel308a K51L protein is unable to unwind fork-2 (lanes g–l), compared with wild-type protein (lanes a–f). Reactions were for 20 min at 45°C containing 2 nM DNA, 5 mM MgCl 2 , 5 mM ATP and 0, 1, 5, 10, 25 or 50 nM Hel308a. Binding of wild-type Hel308a (lanes m–o) and K51L Hel308a (lanes p–r) to fork 2 were in reactions for 10 min at 45°C containing 5 mM MgCl 2 , 2 nM fork-2 (labelled on strand 1) and 0, 10 or 100 nM Hel308a protein.

    Techniques Used: Derivative Assay, Electrophoretic Mobility Shift Assay, Binding Assay

    Mth810 is the archaeal orthologue of metazoan Hel308 in sequence and minimal helicase function. ( A ) Cartoon showing common features of Hel308 from archaea (Hel308a), human (hHel308) and the N-terminal domain of human PolQ. Helicase motifs, including the Q-motif ( 53 ), are labelled and the Hel308a sequences are given for motif I and IVa with mutagenized residues in bold and underlined. ( B ) Sequence details in helicase motifs V and VI that confirm Hel308a as a Hel308/Mus308 family rather than a RecQ helicase. The corresponding motif of human BLM helicase is shown for comparison (hBLM). In each motif peculiar residues conserved in Hel308/Mus308 helicases are in bold. Motif IVa is highly conserved in RecQ and Hel308 proteins. Invariant residues are in bold and highly conserved residues are underlined. Aligned with Hel308a, human Hel308 and human PolQ are Hel308 from Caenorhabditis elegans (CeHel308), E.coli RecQ (EcRecQ) and a human RecQ, BLM (HsBLM). ( C ) SDS–PAGE gel (10% acrylamide) showing purified recombinant Hel308a (arrowed) from Methanothermobacter . Marker sizes are given on the left of the panel. ( D ) ATPase activity of Hel308a measured as a function of time in reactions containing no DNA (filled diamond), dsDNA (open square) or ssDNA (open circles). Error bars are derived from the means of three independent experiments. ( E ) Unwinding reactions of Hel308a on 3′-ssDNA-tailed duplex (i), 5′-ssDNA-tailed duplex (ii) and untailed duplex (iii). Reactions were for 20 min at 45°C containing 2 nM DNA, with 32 P-labelled strand indicated by filled circle, 5 mM MgCl 2 , 5 mM ATP and zero (lane a); 1, 5, 10, 25 and 50 nM Hel308a (lanes b–f).
    Figure Legend Snippet: Mth810 is the archaeal orthologue of metazoan Hel308 in sequence and minimal helicase function. ( A ) Cartoon showing common features of Hel308 from archaea (Hel308a), human (hHel308) and the N-terminal domain of human PolQ. Helicase motifs, including the Q-motif ( 53 ), are labelled and the Hel308a sequences are given for motif I and IVa with mutagenized residues in bold and underlined. ( B ) Sequence details in helicase motifs V and VI that confirm Hel308a as a Hel308/Mus308 family rather than a RecQ helicase. The corresponding motif of human BLM helicase is shown for comparison (hBLM). In each motif peculiar residues conserved in Hel308/Mus308 helicases are in bold. Motif IVa is highly conserved in RecQ and Hel308 proteins. Invariant residues are in bold and highly conserved residues are underlined. Aligned with Hel308a, human Hel308 and human PolQ are Hel308 from Caenorhabditis elegans (CeHel308), E.coli RecQ (EcRecQ) and a human RecQ, BLM (HsBLM). ( C ) SDS–PAGE gel (10% acrylamide) showing purified recombinant Hel308a (arrowed) from Methanothermobacter . Marker sizes are given on the left of the panel. ( D ) ATPase activity of Hel308a measured as a function of time in reactions containing no DNA (filled diamond), dsDNA (open square) or ssDNA (open circles). Error bars are derived from the means of three independent experiments. ( E ) Unwinding reactions of Hel308a on 3′-ssDNA-tailed duplex (i), 5′-ssDNA-tailed duplex (ii) and untailed duplex (iii). Reactions were for 20 min at 45°C containing 2 nM DNA, with 32 P-labelled strand indicated by filled circle, 5 mM MgCl 2 , 5 mM ATP and zero (lane a); 1, 5, 10, 25 and 50 nM Hel308a (lanes b–f).

    Techniques Used: Sequencing, SDS Page, Purification, Recombinant, Marker, Activity Assay, Derivative Assay

    Models proposing how Hel308 helicases may function in archaea and metazoans. ( A ) Displacement of the lagging strand by the Hel308 helicase (Hel) domain of PolQ may provide access for the translesion polymerase domain to DNA damage (grey square, e.g. AP-sites) located on the lagging strand template. ( B ) Hel308a/Hel308 (Hel) unwinding of the lagging strand at fork with a compromised leading or lagging strand provides a template for loading of replication restart apparatus, possibly Pol-α-primase (αPri). In each model, translocation of the helicase is indicted by a dotted arrow, away from the fork branchpoint in a 3′–5′ direction with respect to the lagging strand template.
    Figure Legend Snippet: Models proposing how Hel308 helicases may function in archaea and metazoans. ( A ) Displacement of the lagging strand by the Hel308 helicase (Hel) domain of PolQ may provide access for the translesion polymerase domain to DNA damage (grey square, e.g. AP-sites) located on the lagging strand template. ( B ) Hel308a/Hel308 (Hel) unwinding of the lagging strand at fork with a compromised leading or lagging strand provides a template for loading of replication restart apparatus, possibly Pol-α-primase (αPri). In each model, translocation of the helicase is indicted by a dotted arrow, away from the fork branchpoint in a 3′–5′ direction with respect to the lagging strand template.

    Techniques Used: Translocation Assay

    Hel308 from archaea preferentially targets fork DNA for unwinding. ( A ) Gel-retardation binding assays of Hel308a on the substrates in Figure 2E in 1 mM magnesium. Reactions were at 45°C for 10 min and contained 2 nM DNA substrate mixed with 0, 1, 2, 10, 50 and 100 nM Hel308a. ( B ) The same reactions as in (A), but containing an additional 1 mM ATP in the gel, and all buffers. ( C ). Unwinding reactions of Hel308a on flayed duplex (lanes a–f), fork with leading strand only (lanes g–l), fork with lagging strand only (fork-2, lanes m–r), fork with both leading and lagging strands (fork-1, lanes s–x) and Holliday junction (lanes y–dd). Reactions were for 20 min at 45°C containing 2 nM DNA, 5 mM MgCl 2 , 5 mM ATP and zero (lanes a, g, m, s and y) or 1, 5, 10, 25 and 50 nM Hel308a.
    Figure Legend Snippet: Hel308 from archaea preferentially targets fork DNA for unwinding. ( A ) Gel-retardation binding assays of Hel308a on the substrates in Figure 2E in 1 mM magnesium. Reactions were at 45°C for 10 min and contained 2 nM DNA substrate mixed with 0, 1, 2, 10, 50 and 100 nM Hel308a. ( B ) The same reactions as in (A), but containing an additional 1 mM ATP in the gel, and all buffers. ( C ). Unwinding reactions of Hel308a on flayed duplex (lanes a–f), fork with leading strand only (lanes g–l), fork with lagging strand only (fork-2, lanes m–r), fork with both leading and lagging strands (fork-1, lanes s–x) and Holliday junction (lanes y–dd). Reactions were for 20 min at 45°C containing 2 nM DNA, 5 mM MgCl 2 , 5 mM ATP and zero (lanes a, g, m, s and y) or 1, 5, 10, 25 and 50 nM Hel308a.

    Techniques Used: Electrophoretic Mobility Shift Assay, Binding Assay

    Hel308a unwinds lagging strands from nicks and the fork branchpoint. ( A ) Products from unwinding fork-2 by hel308a as a function of time. Reactions contained 2 nM DNA and 20 nM Hel308a in 5 mM MgCl 2 , 5 mM ATP at 45°C. ( B ) Reactions showing unwinding of strands in fork-1 by Hel308a. Cartoons of the substrate are shown above the panel in each case with the labelled strand denoted by a filled circle. Strands are numbered on one of these cartoons. Reactions were at 45°C for 20 min containing 5 mM MgCl 2 , 5 mM ATP, 2 nM DNA and zero (0) or 50 nM Hel308a (H). Lanes marked B contained no Hel308a and the reactions were heated to 95°C for 20 min. Letters X, Y and Z highlight the major product of unwinding in each reaction containing Hel308a. Substrate markers corresponding to fork lacking a lagging strand (M1), flayed duplex (M2) and partial duplex (M3) are annotated beside the panel. ( C ) Time-course unwinding of fork-3 and its corresponding nicked duplex DNA substrate. Substrates used are annotated to the right of the graph. Reactions were at 45°C for the times shown and contained 2 nM DNA, 5 mM MgCl 2 , 5 mM ATP and 20 nM Hel308a. Error bars derive from means of two independent assays. ( D ) Unwinding of the invading strand of D-loop substrates by Hel308a. The D-loop substrates used are annotated above the panel and reactions products are displayed to the right of the panel. Reactions were for 20 min at 45°C containing 2 nM DNA, 5 mM MgCl 2 , 5 mM ATP and zero or 1, 5, 10, 25 and 50 nM Hel308a.
    Figure Legend Snippet: Hel308a unwinds lagging strands from nicks and the fork branchpoint. ( A ) Products from unwinding fork-2 by hel308a as a function of time. Reactions contained 2 nM DNA and 20 nM Hel308a in 5 mM MgCl 2 , 5 mM ATP at 45°C. ( B ) Reactions showing unwinding of strands in fork-1 by Hel308a. Cartoons of the substrate are shown above the panel in each case with the labelled strand denoted by a filled circle. Strands are numbered on one of these cartoons. Reactions were at 45°C for 20 min containing 5 mM MgCl 2 , 5 mM ATP, 2 nM DNA and zero (0) or 50 nM Hel308a (H). Lanes marked B contained no Hel308a and the reactions were heated to 95°C for 20 min. Letters X, Y and Z highlight the major product of unwinding in each reaction containing Hel308a. Substrate markers corresponding to fork lacking a lagging strand (M1), flayed duplex (M2) and partial duplex (M3) are annotated beside the panel. ( C ) Time-course unwinding of fork-3 and its corresponding nicked duplex DNA substrate. Substrates used are annotated to the right of the graph. Reactions were at 45°C for the times shown and contained 2 nM DNA, 5 mM MgCl 2 , 5 mM ATP and 20 nM Hel308a. Error bars derive from means of two independent assays. ( D ) Unwinding of the invading strand of D-loop substrates by Hel308a. The D-loop substrates used are annotated above the panel and reactions products are displayed to the right of the panel. Reactions were for 20 min at 45°C containing 2 nM DNA, 5 mM MgCl 2 , 5 mM ATP and zero or 1, 5, 10, 25 and 50 nM Hel308a.

    Techniques Used:

    3) Product Images from "Archaeal Hel308 helicase targets replication forks in vivo and in vitro and unwinds lagging strands"

    Article Title: Archaeal Hel308 helicase targets replication forks in vivo and in vitro and unwinds lagging strands

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki685

    Mth810 is the archaeal orthologue of metazoan Hel308 in sequence and minimal helicase function. ( A ) Cartoon showing common features of Hel308 from archaea (Hel308a), human (hHel308) and the N-terminal domain of human PolQ. Helicase motifs, including the Q-motif ( 53 ), are labelled and the Hel308a sequences are given for motif I and IVa with mutagenized residues in bold and underlined. ( B ) Sequence details in helicase motifs V and VI that confirm Hel308a as a Hel308/Mus308 family rather than a RecQ helicase. The corresponding motif of human BLM helicase is shown for comparison (hBLM). In each motif peculiar residues conserved in Hel308/Mus308 helicases are in bold. Motif IVa is highly conserved in RecQ and Hel308 proteins. Invariant residues are in bold and highly conserved residues are underlined. Aligned with Hel308a, human Hel308 and human PolQ are Hel308 from Caenorhabditis elegans (CeHel308), E.coli RecQ (EcRecQ) and a human RecQ, BLM (HsBLM). ( C ) SDS–PAGE gel (10% acrylamide) showing purified recombinant Hel308a (arrowed) from Methanothermobacter . Marker sizes are given on the left of the panel. ( D ) ATPase activity of Hel308a measured as a function of time in reactions containing no DNA (filled diamond), dsDNA (open square) or ssDNA (open circles). Error bars are derived from the means of three independent experiments. ( E ) Unwinding reactions of Hel308a on 3′-ssDNA-tailed duplex (i), 5′-ssDNA-tailed duplex (ii) and untailed duplex (iii). Reactions were for 20 min at 45°C containing 2 nM DNA, with 32 P-labelled strand indicated by filled circle, 5 mM MgCl 2 , 5 mM ATP and zero (lane a); 1, 5, 10, 25 and 50 nM Hel308a (lanes b–f).
    Figure Legend Snippet: Mth810 is the archaeal orthologue of metazoan Hel308 in sequence and minimal helicase function. ( A ) Cartoon showing common features of Hel308 from archaea (Hel308a), human (hHel308) and the N-terminal domain of human PolQ. Helicase motifs, including the Q-motif ( 53 ), are labelled and the Hel308a sequences are given for motif I and IVa with mutagenized residues in bold and underlined. ( B ) Sequence details in helicase motifs V and VI that confirm Hel308a as a Hel308/Mus308 family rather than a RecQ helicase. The corresponding motif of human BLM helicase is shown for comparison (hBLM). In each motif peculiar residues conserved in Hel308/Mus308 helicases are in bold. Motif IVa is highly conserved in RecQ and Hel308 proteins. Invariant residues are in bold and highly conserved residues are underlined. Aligned with Hel308a, human Hel308 and human PolQ are Hel308 from Caenorhabditis elegans (CeHel308), E.coli RecQ (EcRecQ) and a human RecQ, BLM (HsBLM). ( C ) SDS–PAGE gel (10% acrylamide) showing purified recombinant Hel308a (arrowed) from Methanothermobacter . Marker sizes are given on the left of the panel. ( D ) ATPase activity of Hel308a measured as a function of time in reactions containing no DNA (filled diamond), dsDNA (open square) or ssDNA (open circles). Error bars are derived from the means of three independent experiments. ( E ) Unwinding reactions of Hel308a on 3′-ssDNA-tailed duplex (i), 5′-ssDNA-tailed duplex (ii) and untailed duplex (iii). Reactions were for 20 min at 45°C containing 2 nM DNA, with 32 P-labelled strand indicated by filled circle, 5 mM MgCl 2 , 5 mM ATP and zero (lane a); 1, 5, 10, 25 and 50 nM Hel308a (lanes b–f).

    Techniques Used: Sequencing, SDS Page, Purification, Recombinant, Marker, Activity Assay, Derivative Assay

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    New England Biolabs rfii circular double stranded dna dsdna substrates
    Mth810 is the archaeal orthologue of metazoan Hel308 in sequence and minimal helicase function. ( A ) Cartoon showing common features of Hel308 from archaea (Hel308a), human (hHel308) and the N-terminal domain of human PolQ. Helicase motifs, including the Q-motif ( 53 ), are labelled and the Hel308a sequences are given for motif I and IVa with mutagenized residues in bold and underlined. ( B ) Sequence details in helicase motifs V and VI that confirm Hel308a as a Hel308/Mus308 family rather than a RecQ helicase. The corresponding motif of human BLM helicase is shown for comparison (hBLM). In each motif peculiar residues conserved in Hel308/Mus308 helicases are in bold. Motif IVa is highly conserved in RecQ and Hel308 proteins. Invariant residues are in bold and highly conserved residues are underlined. Aligned with Hel308a, human Hel308 and human PolQ are Hel308 from Caenorhabditis elegans (CeHel308), E.coli RecQ (EcRecQ) and a human RecQ, BLM (HsBLM). ( C ) SDS–PAGE gel (10% acrylamide) showing purified recombinant Hel308a (arrowed) from Methanothermobacter . Marker sizes are given on the left of the panel. ( D ) ATPase activity of Hel308a measured as a function of time in reactions containing no <t>DNA</t> (filled diamond), <t>dsDNA</t> (open square) or ssDNA (open circles). Error bars are derived from the means of three independent experiments. ( E ) Unwinding reactions of Hel308a on 3′-ssDNA-tailed duplex (i), 5′-ssDNA-tailed duplex (ii) and untailed duplex (iii). Reactions were for 20 min at 45°C containing 2 nM DNA, with 32 P-labelled strand indicated by filled circle, 5 mM MgCl 2 , 5 mM ATP and zero (lane a); 1, 5, 10, 25 and 50 nM Hel308a (lanes b–f).
    Rfii Circular Double Stranded Dna Dsdna Substrates, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rfii circular double stranded dna dsdna substrates/product/New England Biolabs
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Mth810 is the archaeal orthologue of metazoan Hel308 in sequence and minimal helicase function. ( A ) Cartoon showing common features of Hel308 from archaea (Hel308a), human (hHel308) and the N-terminal domain of human PolQ. Helicase motifs, including the Q-motif ( 53 ), are labelled and the Hel308a sequences are given for motif I and IVa with mutagenized residues in bold and underlined. ( B ) Sequence details in helicase motifs V and VI that confirm Hel308a as a Hel308/Mus308 family rather than a RecQ helicase. The corresponding motif of human BLM helicase is shown for comparison (hBLM). In each motif peculiar residues conserved in Hel308/Mus308 helicases are in bold. Motif IVa is highly conserved in RecQ and Hel308 proteins. Invariant residues are in bold and highly conserved residues are underlined. Aligned with Hel308a, human Hel308 and human PolQ are Hel308 from Caenorhabditis elegans (CeHel308), E.coli RecQ (EcRecQ) and a human RecQ, BLM (HsBLM). ( C ) SDS–PAGE gel (10% acrylamide) showing purified recombinant Hel308a (arrowed) from Methanothermobacter . Marker sizes are given on the left of the panel. ( D ) ATPase activity of Hel308a measured as a function of time in reactions containing no DNA (filled diamond), dsDNA (open square) or ssDNA (open circles). Error bars are derived from the means of three independent experiments. ( E ) Unwinding reactions of Hel308a on 3′-ssDNA-tailed duplex (i), 5′-ssDNA-tailed duplex (ii) and untailed duplex (iii). Reactions were for 20 min at 45°C containing 2 nM DNA, with 32 P-labelled strand indicated by filled circle, 5 mM MgCl 2 , 5 mM ATP and zero (lane a); 1, 5, 10, 25 and 50 nM Hel308a (lanes b–f).

    Journal: Nucleic Acids Research

    Article Title: Archaeal Hel308 helicase targets replication forks in vivo and in vitro and unwinds lagging strands

    doi: 10.1093/nar/gki685

    Figure Lengend Snippet: Mth810 is the archaeal orthologue of metazoan Hel308 in sequence and minimal helicase function. ( A ) Cartoon showing common features of Hel308 from archaea (Hel308a), human (hHel308) and the N-terminal domain of human PolQ. Helicase motifs, including the Q-motif ( 53 ), are labelled and the Hel308a sequences are given for motif I and IVa with mutagenized residues in bold and underlined. ( B ) Sequence details in helicase motifs V and VI that confirm Hel308a as a Hel308/Mus308 family rather than a RecQ helicase. The corresponding motif of human BLM helicase is shown for comparison (hBLM). In each motif peculiar residues conserved in Hel308/Mus308 helicases are in bold. Motif IVa is highly conserved in RecQ and Hel308 proteins. Invariant residues are in bold and highly conserved residues are underlined. Aligned with Hel308a, human Hel308 and human PolQ are Hel308 from Caenorhabditis elegans (CeHel308), E.coli RecQ (EcRecQ) and a human RecQ, BLM (HsBLM). ( C ) SDS–PAGE gel (10% acrylamide) showing purified recombinant Hel308a (arrowed) from Methanothermobacter . Marker sizes are given on the left of the panel. ( D ) ATPase activity of Hel308a measured as a function of time in reactions containing no DNA (filled diamond), dsDNA (open square) or ssDNA (open circles). Error bars are derived from the means of three independent experiments. ( E ) Unwinding reactions of Hel308a on 3′-ssDNA-tailed duplex (i), 5′-ssDNA-tailed duplex (ii) and untailed duplex (iii). Reactions were for 20 min at 45°C containing 2 nM DNA, with 32 P-labelled strand indicated by filled circle, 5 mM MgCl 2 , 5 mM ATP and zero (lane a); 1, 5, 10, 25 and 50 nM Hel308a (lanes b–f).

    Article Snippet: ATPase assays Hydrolysis of ATP by Hel308a was measured spectroscopically using malachite green assays ( ). φX174 circular ssDNA and RFII circular double-stranded DNA (dsDNA) substrates were from New England Biolabs.

    Techniques: Sequencing, SDS Page, Purification, Recombinant, Marker, Activity Assay, Derivative Assay