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Proteins that act at specific DNA sequences bind DNA randomly and then …


Biology Articles » Biophysics » Molecular Biophysics » Measurement of the contributions of 1D and 3D pathways to the translocation of a protein along DNA » Materials and methods

Materials and methods
- Measurement of the contributions of 1D and 3D pathways to the translocation of a protein along DNA

Enzymes. The BbvCI restriction enzyme, and its M1.gifequation M1 mutant, were purified as described in ref. 30, and their concentrations, in terms of the Mr 64053 R1R2 heterodimer, were determined from A280 readings (31). Other enzymes were from New England Biolabs.

DNA. DNA manipulations were performed according to standard procedures (32) and checked by sequencing (University of Dundee, Dundee, U.K.). A duplex with the recognition sequence for BbvCI (underlined), was made from the oligonucleotides

M2.gif

(MWG Biotech, Ebersberg, Germany) and ligated to EcoRI-cut pUC19 (33). One recombinant contained a single insert in the above orientation (i.e., with the CCTCAGC sequence in the “top” strand). Further derivatives were constructed from this plasmid by inserting into its polylinker a second duplex with a BbvCI site at the SacI, AvaI, BamHI, or HindIII site. For example, the duplex
M3.gif 

was cloned at the HindIII site to yield recombinants with two BbvCI sites 75 bp apart. Some had the insert in the same orientation as above (with CCTCAGC again in the top strand), whereas others had the inverted orientation (with CCTCAGC in the “bottom” strand). The duplexes inserted at the SacI, AvaI, and BamHI sites had the same sequence as the HindIII insert, apart from the “sticky ends,” and gave both repeated and inverted derivatives with two BbvCI sites separated by, respectively, 30, 40, and 45 bp.

For each plasmid, a 301-bp section that spanned the two BbvCI sites was amplified by PCR. The reactions contained 0.1 nM plasmid, 1 μM top and bottom strand primers, 0.37 MBq α32P-dATP (GE Healthcare, Chalfont St. Giles, U.K.), and PCR Master Mix (Promega). The two primers matched, respectively, loci 307–326 and 562–543 in pUC19 (33). The products were recovered with a PCR purification kit (Qiagen, West Sussex, U.K.), and a sample of each was analyzed on agarose gels alongside quantitative GIBCO/BRL markers. Their concentrations were determined by reference to the intensities of the markers (Kodak imagestation 1d software).

Reactions. Solutions at 37°C contained 30 nM 32P-labeled DNA (one of the above PCR fragments) in 100 μl of Reaction Buffer (10 mM Tris·HCl/5 mM MgCl2/1 mM 2-mercaptoethanol/100 μg/ml BSA, pH 7.4), sometimes supplemented with 30, 60, or 150 mM NaCl. An aliquot (5 μl) was taken at zero time and mixed with 10 μl of Stop Mix (26) before adding to the remainder BbvCI (diluted as described in ref. 31) to 0.3 nM. At intervals thereafter, further aliquots were taken, mixed immediately with Stop Mix, and subjected to electrophoresis through 8% polyacrylamide in TBE (32), in water-cooled 20-cm gels at 40 mA for 3 h. Gels were dried, exposed to a Molecular Dynamics phosphorimager, and analyzed in imagequant (Molecular Dynamics) to assess the amounts of each DNA in each sample. The total counts in rectangles encompassing each band were summed and corrected for local background, and the values were converted to DNA concentrations by reference to the number of adenines in the fragment (α32P-dATP was used in the PCR). Initial rates were evaluated from the first 15% of the reaction with grafit (Erithacus Software, Surrey, U.K.).

Nonspecific Binding. Varied concentrations of BbvCI (0–25 μM) were added to a 21-bp duplex (30 nM, 32P-labeled with kinase; ref. 32) in Reaction Buffer (except for CaCl2 instead of MgCl2) with NaCl as indicated. The duplex had the same sequence as those above, except that the BbvCI recognition sequence was replaced by the reverse, 5′-CGACTCC-3′. The fraction of the duplex bound to BbvCI was assessed by gel-retardation (34), and a KD for nonspecific binding was evaluated from the decline in this fraction with increasing [E0] (35).


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