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Mapping DNA cleavage by the Type ISP restriction-modification enzymes following long-range communication between DNA sites in different orientations.

van Aelst K, Saikrishnan K, Szczelkun MD - Nucleic Acids Res. (2015)

Bottom Line: By following communication between sites in both head-to-head and head-to-tail orientations, we could show that motor activity leads to activation of the nuclease domains via distant interactions of the helicase or MTase-TRD.Direct nuclease dimerization is not required.To help explain the observed cleavage patterns, we also used exonuclease footprinting to demonstrate that individual Type ISP domains can swing off the DNA.

View Article: PubMed Central - PubMed

Affiliation: DNA-Protein Interactions Unit, School of Biochemistry, University of Bristol, Bristol BS8 1TD, UK.

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Cleavage of head-to-tail DNA results from rear-end collision between a translocating enzyme and an enzyme at a site. (A) Cartoon of the linear DNA substrate. The arrows show the orientations of the LlaBIII (blue) and LlaGI (red) targets, where the arrowheads indicate the direction of translocation (viz. Figure 1A). Type II restriction enzyme positions are given for the nucleotide 5′ to the phosphodiester cleaved on the top strand (according to standard definitions (5)). DNA (5 nM), 5′-labeled on either the top strand (B) or bottom strand (C) with 32-phosphorus (sun symbols), was incubated with the enzymes shown (200 nM of each) for 2 min. ATP was added to 4 mM, the reaction incubated for 10 min at 25°C and then stopped. Products were separated by alkaline denaturing agarose gel electrophoresis. The size of each marker represents the length of the labeled ssDNA produced. Table key: No enzyme (−); WT enzyme (+); nuclease point mutant (N−); nuclease domain deletion (ΔN); or, ATPase point mutant (H−). Gels shown are representative examples from two repeat reactions.
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Figure 3: Cleavage of head-to-tail DNA results from rear-end collision between a translocating enzyme and an enzyme at a site. (A) Cartoon of the linear DNA substrate. The arrows show the orientations of the LlaBIII (blue) and LlaGI (red) targets, where the arrowheads indicate the direction of translocation (viz. Figure 1A). Type II restriction enzyme positions are given for the nucleotide 5′ to the phosphodiester cleaved on the top strand (according to standard definitions (5)). DNA (5 nM), 5′-labeled on either the top strand (B) or bottom strand (C) with 32-phosphorus (sun symbols), was incubated with the enzymes shown (200 nM of each) for 2 min. ATP was added to 4 mM, the reaction incubated for 10 min at 25°C and then stopped. Products were separated by alkaline denaturing agarose gel electrophoresis. The size of each marker represents the length of the labeled ssDNA produced. Table key: No enzyme (−); WT enzyme (+); nuclease point mutant (N−); nuclease domain deletion (ΔN); or, ATPase point mutant (H−). Gels shown are representative examples from two repeat reactions.

Mentions: The linear DNA for the mapping and footprinting experiments were generated by PCR, with either the forward or reverse primer being 32P-labeled at the 5′ end using T4 polynucleotide kinase and γ32P-ATP by standard techniques (15). The 1188 bp linear HtT mixed DNA (Figures 3 and 4) was generated from the 1739-240 region of pUC19 (16) using primers KA084F (5′-CTGGCCCCAGTGCTGCAATGATAC-3′) and KA084R (5′-GGCGCCTGATGCGGTATTTTCTC-3′). The 256 bp linear HtH mixed DNA (Figure 5) was generated from the 6–261 region of pEX-A-KA1 (see below) using primers F80 (5′-CACGATGAAGAACTATCTGCTTCCGATTGTG-3′) and R80 (5′-ATTATGGGTTTGTTGCACGGGTTGGTC-3′).


Mapping DNA cleavage by the Type ISP restriction-modification enzymes following long-range communication between DNA sites in different orientations.

van Aelst K, Saikrishnan K, Szczelkun MD - Nucleic Acids Res. (2015)

Cleavage of head-to-tail DNA results from rear-end collision between a translocating enzyme and an enzyme at a site. (A) Cartoon of the linear DNA substrate. The arrows show the orientations of the LlaBIII (blue) and LlaGI (red) targets, where the arrowheads indicate the direction of translocation (viz. Figure 1A). Type II restriction enzyme positions are given for the nucleotide 5′ to the phosphodiester cleaved on the top strand (according to standard definitions (5)). DNA (5 nM), 5′-labeled on either the top strand (B) or bottom strand (C) with 32-phosphorus (sun symbols), was incubated with the enzymes shown (200 nM of each) for 2 min. ATP was added to 4 mM, the reaction incubated for 10 min at 25°C and then stopped. Products were separated by alkaline denaturing agarose gel electrophoresis. The size of each marker represents the length of the labeled ssDNA produced. Table key: No enzyme (−); WT enzyme (+); nuclease point mutant (N−); nuclease domain deletion (ΔN); or, ATPase point mutant (H−). Gels shown are representative examples from two repeat reactions.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4666363&req=5

Figure 3: Cleavage of head-to-tail DNA results from rear-end collision between a translocating enzyme and an enzyme at a site. (A) Cartoon of the linear DNA substrate. The arrows show the orientations of the LlaBIII (blue) and LlaGI (red) targets, where the arrowheads indicate the direction of translocation (viz. Figure 1A). Type II restriction enzyme positions are given for the nucleotide 5′ to the phosphodiester cleaved on the top strand (according to standard definitions (5)). DNA (5 nM), 5′-labeled on either the top strand (B) or bottom strand (C) with 32-phosphorus (sun symbols), was incubated with the enzymes shown (200 nM of each) for 2 min. ATP was added to 4 mM, the reaction incubated for 10 min at 25°C and then stopped. Products were separated by alkaline denaturing agarose gel electrophoresis. The size of each marker represents the length of the labeled ssDNA produced. Table key: No enzyme (−); WT enzyme (+); nuclease point mutant (N−); nuclease domain deletion (ΔN); or, ATPase point mutant (H−). Gels shown are representative examples from two repeat reactions.
Mentions: The linear DNA for the mapping and footprinting experiments were generated by PCR, with either the forward or reverse primer being 32P-labeled at the 5′ end using T4 polynucleotide kinase and γ32P-ATP by standard techniques (15). The 1188 bp linear HtT mixed DNA (Figures 3 and 4) was generated from the 1739-240 region of pUC19 (16) using primers KA084F (5′-CTGGCCCCAGTGCTGCAATGATAC-3′) and KA084R (5′-GGCGCCTGATGCGGTATTTTCTC-3′). The 256 bp linear HtH mixed DNA (Figure 5) was generated from the 6–261 region of pEX-A-KA1 (see below) using primers F80 (5′-CACGATGAAGAACTATCTGCTTCCGATTGTG-3′) and R80 (5′-ATTATGGGTTTGTTGCACGGGTTGGTC-3′).

Bottom Line: By following communication between sites in both head-to-head and head-to-tail orientations, we could show that motor activity leads to activation of the nuclease domains via distant interactions of the helicase or MTase-TRD.Direct nuclease dimerization is not required.To help explain the observed cleavage patterns, we also used exonuclease footprinting to demonstrate that individual Type ISP domains can swing off the DNA.

View Article: PubMed Central - PubMed

Affiliation: DNA-Protein Interactions Unit, School of Biochemistry, University of Bristol, Bristol BS8 1TD, UK.

Show MeSH
Related in: MedlinePlus