Example 1-Making an extended 4 way junction.

It has been shown that in low concentrations of metal ions, the 4-way Holliday junction forms an extended structure. (Duckett, et al; Clegg, et al).

The secondary structure:

      GC
      CG
      GC
      CG
      GC
      CG
GCGCGC  AGAGAG
CGCGCG  TCTCTC
      AT
      GC
      AT
      GC
      AT
      GC
To construct the junction:
  1. Add an AT unit( Dx:0.0; Dy:0.0; Dz:0.0; Tilt:0.0; Twist:0.0; Roll:0.0). The Helix,Unit, Attachements, and Strand direction fields should be set to zero. See figure 1.
  2. Add an AT unit( Dx:0.0; Dy:10.0; Dz:12.0; Tilt:-90.0; Twist:0.0; Roll:0.0). The helix field should contain "2". The Attachements should be "1:1:2 1:-1". The remaining fields should be zero. See figure 2.
  3. Add a CG unit( Dx:0.0; Dy:0.0; Dz:23.0; Tilt:0.0; Twist:0.0; Roll:0.0). The helix field should contain "3". The Attachements should be "1:3:2 1:-1". The remaining fields should be remain zero. See figure 3.
  4. Add a AT unit( Dx:0.0; Dy:-10.0; Dz:12.0; Tilt:90.0; Twist:0.0; Roll:0.0). The helix field should contain "4". The Attachements should be "1:4:2 1:2:0". The remaining fields can remain zero. See figure 4.
To construct the arms:
  1. The first arm is the hardest to build. The "AT" of the first helix that is involved in the junction is the last of the units in the helix. In order to keep the position of the final "AT" the same, the first unit's parameters will have to be set correctly.
    1. Add the units in B-form.
    2. the helix and unit fields will be "1" for all the units added for this strand.
    3. The first entry in the Attachements field will be "1:1:0". The second entry will always be the last group in the second strand plue one. (i.e.: for the first unit to be added the entire field will be:"1:1:0 1:2:3")
    4. Once the units are added, the position of the first unit of the helix should be corrected so that the final "AT" pair is in the correct position. To do this, apply a -20.4(6*-3.4) angstrom change in Dz, and a -204(6*-34) degree change in twist to unit 1:1. The parameters for unit "6" will need to be changed to Dx:0.0; Dy:0.0 Dz:3.4; Tilt:0.0; Twist:34.0; Tilt:0.0. NOTE: if using a NAMOT2 version from Dec 19 or later, this is unneccessary. The program will do this for the user.
    See figure 5.
  2. Completing the second arm is straight forward
    1. Set the helix field to "2", the unit entry will range from 2-6 as you add the units.
    2. The first Attachements entry will range from 1:1:8 to 1:1:12, the second entry will remain 1:3:0.
    3. Add the units in B-form.
    See figure 6.
  3. Add the third and fourth arms the same way as the second arm. See figure 7. See figure 8.
For clarity, a script is provided.

References

Duckett G.R., Murchie A.I.H., Clegg, R.M., Zechel, A., von Kitizing E., Diekmann. S. and Lilley, D.M.J. The Structure of the Holliday Junction. Sarma Rh and Sarma MH(eds) Structure and Methods Vol 1:Human Genome Initiative & DNA Recombination. Adenine Press, New York 157-182

Clegg R.M., Murchie A.I., Lilley D.M. Biophys J 66: 99-109 (1994)