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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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Related in: MedlinePlus

Mapping the cleavage events on the bottom strand following rear-end collision at a site. (A) Cartoon of the linear DNA substrate from Figure 3. The sequence of the LlaGI site is highlighted, along with the locations cleaved by LlaBIII. The sun symbol shows the labeling with 32-phosphorus. (B) DNA (2 nM), 5′-labeled on the bottom strand 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. The products were separated by denaturing polyacrylamide gel electrophoresis. (C) Quantified band intensities for lanes 6–13 (in grey) in panel B (Materials and Methods). Bar represents 500 intensity units. Tables key: No enzyme (−); WT enzyme (+ or WT); nuclease point mutant (N−); nuclease domain deletion (ΔN); or, ATPase point mutant (H−). Gel and quantitation shown are representative examples from two repeat reactions.
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Figure 4: Mapping the cleavage events on the bottom strand following rear-end collision at a site. (A) Cartoon of the linear DNA substrate from Figure 3. The sequence of the LlaGI site is highlighted, along with the locations cleaved by LlaBIII. The sun symbol shows the labeling with 32-phosphorus. (B) DNA (2 nM), 5′-labeled on the bottom strand 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. The products were separated by denaturing polyacrylamide gel electrophoresis. (C) Quantified band intensities for lanes 6–13 (in grey) in panel B (Materials and Methods). Bar represents 500 intensity units. Tables key: No enzyme (−); WT enzyme (+ or WT); nuclease point mutant (N−); nuclease domain deletion (ΔN); or, ATPase point mutant (H−). Gel and quantitation 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)

Mapping the cleavage events on the bottom strand following rear-end collision at a site. (A) Cartoon of the linear DNA substrate from Figure 3. The sequence of the LlaGI site is highlighted, along with the locations cleaved by LlaBIII. The sun symbol shows the labeling with 32-phosphorus. (B) DNA (2 nM), 5′-labeled on the bottom strand 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. The products were separated by denaturing polyacrylamide gel electrophoresis. (C) Quantified band intensities for lanes 6–13 (in grey) in panel B (Materials and Methods). Bar represents 500 intensity units. Tables key: No enzyme (−); WT enzyme (+ or WT); nuclease point mutant (N−); nuclease domain deletion (ΔN); or, ATPase point mutant (H−). Gel and quantitation shown are representative examples from two repeat reactions.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4666363&req=5

Figure 4: Mapping the cleavage events on the bottom strand following rear-end collision at a site. (A) Cartoon of the linear DNA substrate from Figure 3. The sequence of the LlaGI site is highlighted, along with the locations cleaved by LlaBIII. The sun symbol shows the labeling with 32-phosphorus. (B) DNA (2 nM), 5′-labeled on the bottom strand 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. The products were separated by denaturing polyacrylamide gel electrophoresis. (C) Quantified band intensities for lanes 6–13 (in grey) in panel B (Materials and Methods). Bar represents 500 intensity units. Tables key: No enzyme (−); WT enzyme (+ or WT); nuclease point mutant (N−); nuclease domain deletion (ΔN); or, ATPase point mutant (H−). Gel and quantitation 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