C581 Problem Set #2

Names _________________

• Login using "C581" as the user, and type in the password supplied in class.
• Open a UNIX shell and change to your directory (cd lastname_firstname).
• Start Insight2.

It will really help to have a copy of the text in front of you while working through this problem set.

Triplex DNA

1. Now, you are going to build you own Triplex DNA helix.
• Click on the blue and white star-like icon in the upper left corner.
• Select the "Biopolymer" option. A second row of commands should appear at the top of the screen.
• Choose "Nucleotide" and then "Append."
• Make sure the A_DNA_Triplex is selected.
• Name your molecule in the "Molecule Name" box.
• Next, click on the "Nucleotide" box. You are only allowed TAT. Build a molecule by clicking on this ~10 times. Besides TAT, what is another common triple base pair?

If it is easier to view the molecule, you can color the molecule using the "colat" command.

2. Color the homopurine strand blue, the homopyrimidine strand yellow, and the Hoogsteen strand red. To do this, click on "Molecule" and select "Color. " Make sure "Atoms" is selected in the Attribute box. Select Triple_1, Triple_2, or Triple_3 depending on which strand you would like to color. Click in the "Color" box and select the appropriate color. Click on "Execute."

Save this file in your directory by selecting "File" and then " Save_Folder." Click in the "Folder Name" box and type the name of the file. Click on "Execute." Indicate below whose directory the file is saved in and the name of the file.

Which bases are the pyrimidines and which are the purines? How can you tell the difference between the Hoogsteen and the homopyrimidine strand?

3. You can also draw a ribbon through the DNA backbone. To do this, click on "Molecule" and select "Ribbon." Make sure "Create" is selected in the Ribbon Operation box and "Nucleic" is selected in the Molecular Type box. Select Triple_1, Triple_2, or Triple _3 depending on which strand you would like to ribbon. Click on "Execute."
4. Draw a schematic representation of the triplex (lines or arrows, but with the 5’ or 3’ ends clearly labeled).
5. Draw a base triplet below. Indicate the hydrogen bonds.
6. Without the third strand, which form of DNA does the remainder of the triplex most resemble? How do you know? (Hint: Consider the groove widths, sugar pucker, etc.) Does the third strand wrap through the major or minor groove?

Delete this molecule.

G Quartet

Click on "Molecule" and select "Get." Make sure "PDB" is selected in the Get File Type box and "User" is selected in the PDB Directory box. Click on "../" in the Files box. Select dterwey/ and choose 581 acids/. Finally, select the file GquartetEDIT.pdb.

1. Last time we learned to select how much of the molecule was viewed by clicking on the side view box and moving the bars to select the desired view. There is another way to select the layer size. Do this by clicking on "Slab Thick" on the bottom of the screen. In the lower left corner there will be a box that says slab thick in it. By clicking on it with the left mouse button, you can select the layer size in angstroms. Clicking on the left side of the box increases the slab thickness and clicking on the right side of the box decreases the slab thickness. Set this to approximately 50 angstroms.
2. Draw a simple sketch of the G tetraplex. This will be easier if you draw a ribbon along the backbone. To do this, click on "Molecule" and select "Ribbon." Make sure "Nucleic" is selected in the Molecule Type box and click on "Execute." Redraw this backbone below indicating the 5’ and 3’ ends. Are the two strands parallel or antiparallel?
3. Rotate the molecule around until you view a structure containing three flat planes in the center of the structure and loops on two sides. The planes are very flat and have a large empty space between them. These flat planes are the G quartet. You will need to view a single G quartet. Use can either set the slab thickness to ~5 angstroms or do the following, whichever is easiest for you. To do this, select all the atoms above the G quartet (Use the left mouse button to draw a box around these atoms. When you release the mouse button, the atoms will be selected. Hit the delete key to get rid of these atoms. This will take a minute or so.). Repeat this for the atoms below the G quartet. Describe the hydrogen-bonding pattern in the G quartet. Draw a G quartet and indicate the hydrogen bonds. Measure the dihedral angle for each guanosine in the quartet. Are they in the syn or anti conformation? Use the back of the page if you need more room.

A Tracts

We will look at a helix with a run of A:T base pairs. This structure was synthesized with G:C base pairs on the end to hold it together. Therefore, be sure to look at the middle of the molecule when you answer the questions.

Click on "Molecule" and select "Get." Click on "../" in the File box. Let it bring up a menu and then click on "../" again. Choose C581/ and then select slato/. Finally, choose the file Atract.pdb.

1. Measure the distance between N6 of adenine and O4 of thymine. Next, follow the A containing strand down the 3’ side to the next base pair. Measure the distance from the N6 of the adenine you just measured to the O4 of the 3’-thymine in the next base pair. List the distances you measured below.
2. Rotate the molecule so that you can observe the propeller twist. Is it more or less than you observed for B-DNA? Measure any distance that would illustrate this point. Is there anything about the sequence that would explain this?

Delete this molecule.

Intercalators

1. The last molecule for this problem set is a stretch of double stranded DNA bound to an intercalator. What is an intercalator?
2. Open a UNIX shell. Type "jot triostinA.pdb". This will list the contents of this file. Scroll through this file until you get to a section that tell you the contents of the A, B, C, and D strands for this molecule. What do the A, B, C, and D strands represent?
3. Exit the file and UNIX shell. Go to "File" and select " Restore_Folder." Choose triostinA.psv. Rotate this molecule so that you can see the quinoxaline rings of the intercalator sticking through the DNA. TriostinA is a dimer linked through a disulfide bond. The monomer contains a quinoxaline and a 4 amino acid peptide chain. What color is the disulfide bond?
4. Go to "Molecule" and select "Render." Select "CPK" in the Render Style box and TRIOSTINA1 in the Molecular Specs box. Click on "Execute. " Go to "Molecule" and select "Color." Choose TRIOSTINA1 in the Molecular Specs box. This time we only want to color the DNA, not the intercalator. After TRIOSTINA1 type a :X*,Y* where X and Y represent the strands of DNA you determined from jotting the file. Color the DNA light blue or magenta. Click on "Execute." What are the two base pairs sandwiched between the intercalator?
5. Briefly describe the consequence the intercalator has on the normally rise of the DNA base pairs, as well as, the consequence on the overall DNA structure.