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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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Exonuclease III DNA footprinting of a wild type LlaGI-DNA complex in the absence of ATP. (A) DNA (2 nM), 5′-labeled on the top strand (upper gel) or bottom strand (lower gel) with 32-phosphorus was pre-incubated without (−) or with (+) 200 nM LlaGI in the absence of ATP for 2 min. Exonuclease III (to 0.75 U/μl) was added at 25°C and reacted for the times shown. Quenched products were separated by denaturing polyacrylamide gel electrophoresis. (B) Band intensity summated from all time points in panel A (see Materials and Methods and main text for further details). The complete sequence of the 157 bp linear one-site LlaGI DNA is shown, with the PCR primers highlighted in gray. (C) Peak heights for the bands indicated as a function of exonuclease III incubation time. Bars represent 1000 intensity units. Positions are assigned to domains as explained in Figure 8 and the main text. Gels and quantitation shown are representative examples from two repeat reactions.
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Figure 7: Exonuclease III DNA footprinting of a wild type LlaGI-DNA complex in the absence of ATP. (A) DNA (2 nM), 5′-labeled on the top strand (upper gel) or bottom strand (lower gel) with 32-phosphorus was pre-incubated without (−) or with (+) 200 nM LlaGI in the absence of ATP for 2 min. Exonuclease III (to 0.75 U/μl) was added at 25°C and reacted for the times shown. Quenched products were separated by denaturing polyacrylamide gel electrophoresis. (B) Band intensity summated from all time points in panel A (see Materials and Methods and main text for further details). The complete sequence of the 157 bp linear one-site LlaGI DNA is shown, with the PCR primers highlighted in gray. (C) Peak heights for the bands indicated as a function of exonuclease III incubation time. Bars represent 1000 intensity units. Positions are assigned to domains as explained in Figure 8 and the main text. Gels and quantitation shown are representative examples from two repeat reactions.

Mentions: The 157 bp linear one-site LlaGI DNA (Figure 7) was generated from the 1785–1941 region of pUC19 using primers FOli LlaGI +75 (5′-TCCAGATTTATCAGCAATAAACCAGCCAGCC-3′) and ROli LlaGI -75 (5′-AGCAATGGCAACAACGTTGCGCAAACTATTAAC-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)

Exonuclease III DNA footprinting of a wild type LlaGI-DNA complex in the absence of ATP. (A) DNA (2 nM), 5′-labeled on the top strand (upper gel) or bottom strand (lower gel) with 32-phosphorus was pre-incubated without (−) or with (+) 200 nM LlaGI in the absence of ATP for 2 min. Exonuclease III (to 0.75 U/μl) was added at 25°C and reacted for the times shown. Quenched products were separated by denaturing polyacrylamide gel electrophoresis. (B) Band intensity summated from all time points in panel A (see Materials and Methods and main text for further details). The complete sequence of the 157 bp linear one-site LlaGI DNA is shown, with the PCR primers highlighted in gray. (C) Peak heights for the bands indicated as a function of exonuclease III incubation time. Bars represent 1000 intensity units. Positions are assigned to domains as explained in Figure 8 and the main text. Gels 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 7: Exonuclease III DNA footprinting of a wild type LlaGI-DNA complex in the absence of ATP. (A) DNA (2 nM), 5′-labeled on the top strand (upper gel) or bottom strand (lower gel) with 32-phosphorus was pre-incubated without (−) or with (+) 200 nM LlaGI in the absence of ATP for 2 min. Exonuclease III (to 0.75 U/μl) was added at 25°C and reacted for the times shown. Quenched products were separated by denaturing polyacrylamide gel electrophoresis. (B) Band intensity summated from all time points in panel A (see Materials and Methods and main text for further details). The complete sequence of the 157 bp linear one-site LlaGI DNA is shown, with the PCR primers highlighted in gray. (C) Peak heights for the bands indicated as a function of exonuclease III incubation time. Bars represent 1000 intensity units. Positions are assigned to domains as explained in Figure 8 and the main text. Gels and quantitation shown are representative examples from two repeat reactions.
Mentions: The 157 bp linear one-site LlaGI DNA (Figure 7) was generated from the 1785–1941 region of pUC19 using primers FOli LlaGI +75 (5′-TCCAGATTTATCAGCAATAAACCAGCCAGCC-3′) and ROli LlaGI -75 (5′-AGCAATGGCAACAACGTTGCGCAAACTATTAAC-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