Mapping DNA cleavage by the Type ISP restriction-modification enzymes following long-range communication between DNA sites in different orientations.
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.
Affiliation: DNA-Protein Interactions Unit, School of Biochemistry, University of Bristol, Bristol BS8 1TD, UK.Show MeSH
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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′).
Affiliation: DNA-Protein Interactions Unit, School of Biochemistry, University of Bristol, Bristol BS8 1TD, UK.