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Exo III footprinting is a method for determining the site of binding for a protein on a DNA sequence. In order to use this procedure, you must have: purified protein, the DNA sequence of the cloned DNA in question, and filter-binding data indicating the general region of binding (or at least good reason to believe the binding site is within a relatively small region). Before starting the actual exo III footprinting, you need to isolate singley end-labeled probes of both strands containing the binding region, dissolved in ddH2O to 10,000 - 20,000 CPM/ul. These probes should be tested by DMS reactions (use 1/10 of the total amount of each probe - the rest of the DMS-treated probe is used for MW markers during the footprinting) for purity and to be sure the probes are the correct fragments. Labeling at 3' overhangs is OK, but changes in the digestion pattern may occur. 3' overhang second-cuts are NOT allowed - exo III will not reliably digest from the 3' overhang.
Once you have these probes, you must determine the correct amount of exo III required for complete, but not over, digestion of EACH PROBE, empirically by titration. With this information you are ready to begin footprinting assays. Both the titration & footprinting are explained here.
Exo III footprints (i.e.the boundary for the protein nearest to the labeled end) are seen as bands intermediate between the undigested probe and the complete digestion products. Unlike in the exo III titration, where the DNA steadily progresses from undigested to completely digested, with decreasing binding protein the DNA should pass directly from undigested to completely digested, since for each molecule of DNA, either the ends are covered by nonspecific binding or not. Any intermediate band(s) are either footprints or evidence that something in the protein sample is inhibiting the exo III. If the binding protein titration looks like the exo III titration, then it is probably due to inhibition of exo III activity by the protein sample. If only a few intermediate bands occur, they are most likely footprints, especially if they are strong signals.

  1. Materials

    1. Exonuclease III
      Boeringer Mannhiem - 160u/ul Store at -20C.

    2. TMK buffer
      50% glycerol
      50mM Tris pH8
      50mM KCl
      10mM MgCl2

    3. 2X footprint buffer
      100mM KCl [KCl] may be adjusted depending on
      the optimum determined by filter-binding.
      20mM MgCl2
      40mM Tris pH8
      0.2mM EDTA

    4. NTP mix
      3.3mM ATP
      3.3mM GTP
      3.3mM CTP

    5. STOP solution
      0.1M EDTA
      0.6M NH4OAc
      20ug/ml sssDNA

  2. Exonuclease III titration

    1. For each probe, mix (working on ice):
      440ul ddH2O
      50ul 5mg/ml BSA (nuclease-free)
      500ul 2X footprinting buffer
      50ul NTP mix
      10ul probe DNA

    2. Divide the mix into 10 reactions of 100ul each, in eppendorf tubes.
    3. Do 8 1:2 serial dilutions of stock exo III (2ul TMK + 2ul exo III, mix, go to the next dilution).
    4. With all samples starting on ice, the experimental time course is :
      tube # 1 2 3 4 5 6 7 8 9 10
      --> 37C 0min 1 2 3 4 5 6 7 8 9
      Exo III (1ul) 10min 11 12 13 14 15 16 17 18 19
      100ulSTOP->ice 20min 21 22 23 24 25 26 27 28 29
      units exoIII (2ul) 320 160 80 40 20 10 5 2 1 0

    5. When all the samples are done, add 100ul phenol:chloroform to each & vortex. Spin 1-2 min and transfer the upper phases to tubes containing 600ul EtOH. Freeze the samples in a dry-ice:EtOH bath for 5 min, then spin for 5 min. Discard the supernatants, and add 750ul ice-cold 70% EtOH to each tube, refreeze and spin as before.
    6. Resuspend each sample in 5ul sequencing dye and run a 6% sequencing gel. Run an appropriate distance for the size of the DNA used (broomophenol blue to bottom for 150bp, XC to bottom for 400bp). Dry & autoradiograph the gel (overnight exposure with screens at -70C).
    7. Choose the best digest for future use - you want digestion to the half-way point in the DNA, leaving 2 or 3 major bands near the middle of the molecules. Overdigestion results in degradation of the remaining, single-stranded, DNA.
    8. For 3' overhang labeled DNA, complete digestion of the labeled DNA strand may occur (rether than digestion to the half-way point) since exo III will not digest the unlabeled strand from the 3' overhang. In this case, use the lowest concentration of exo III that reliably gives total digestion of the labeled strand.

  3. Footprinting

    1. Working on ice, mix the same reaction mix as before for each probe. Divide the mix into 10 tubes each of 100ul. Prepare a dilution of Exo III if required. You should use the same dilution of exo III that gave optimum digestion in the titration.
    2. Prepare 1:2 dilutions (8 of them) of the binding protein to be tested in TMK. With all samples starting on ice, the time schedule is :
      tube # 1 2 3 4 5 6 7 8 9 10
      1ul protein->37C 0min 1 2 3 4 5 6 7 8 9
      1ul exoIII 10min 11 12 13 14 15 16 17 18 19
      100ul STOP->ice 20min 21 22 23 24 25 26 27 28 29
      [protein] used 1x 1 1 1 1 1 1 1 1 0
      2 4 8 16 32 64 128 256

    3. Phenol:chloroform extract, EtOH precipitate, 70% wash, dry, resuspend & run the 6% sequencing gel as before. Dry the gel and expose as indicated by experience from the exo III titration.
    4. Repeat the footprinting using ddH2O in place of NTP mix.

JWB, unpublished
Shalloway, et al 1980 Cell 20:411-422

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