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LS1a Fall 2014 Additional Practice ICE #2 Questions

Answers are in the back

1. One of the most important features of DNA is that it exists in the cell as a double helix. This provides
the nucleotide sequence with a redundant set of information for error correction and replication. The
double-stranded structure of DNA also enables its recognition by various proteins.

a. List the three factors that contribute to the thermodynamic stability of the DNA double helix.
Which two are the most important?




Proflavin is a chemical that can bind DNA by inserting between base pairs. The chemical structure of
proflavin is drawn below.


An image generated by PyMOL showing proflavin binding to DNA between base pairs is shown below
(green, carbon; red, oxygen; blue, nitrogen; orange, phosphorus; magenta, hydrogen bonds):



b. Identify the four bases in Figure A.
c. On the DNA strand on the right in Figure B, indicate the 3 end and the 5 end.
d. Ethidium and DAPI (shown below) both bind to DNA by inserting between base pairs in the same
manner as proflavin. Naphthalene-2,7-diamine (also shown below), on the other hand, does not
bind DNA. Examine the structures of all four molecules and briefly describe characteristics that
proflavin, ethidium, and DAPI share that allow them to bind to DNA between base pairs. Also
briefly explain why naphtalene-2,7-diamine is unable to bind to DNA between base pairs.



Proflavin
Figure A
Figure B
6.8
2












2. Drawn below are the structures of two DNA base pairs:


On the above diagram:

a. Label each nitrogenous base.
b. Label the major and minor groove of each base pair.
c. Label the patterns of hydrogen bond acceptors, donors and non-polar atoms/groups (all indicated
by dashed circles) presented at the edges of each base pair in the major and minor grooves using
the following nomenclature:
A= Hydrogen bond acceptor
D= Hydrogen bond donor
M= Methyl group (CH3)
H= Hydrogen atom that cannot participate in hydrogen bonds








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3. In addition to the deamination of cytosine, another mutagenic form of DNA damage occurs when the
base guanine is oxidized to become 8-oxo-guanine:

The oxidation of carbon 8 of guanine is mutagenic because 8-oxoguanine can base pair with adenine
instead of cytosine:


a. Why are purine-purine base pairings typically unfavorable?







b. Notice how 8-oxoguanine rotates from its original orientation in order to base pair with adenine.
Why can 8-oxoguanine base pair with adenine in this orientation, but guanine cannot?

















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4. In the space below, do the following:
i. Onto the structure of guanine that is shown below, draw a diagram of how cytosine and guanine
hydrogen bond to one another when base pairing. Use to indicate hydrogen bonds. The
structure of cytosine is shown below.
ii. Circle the atom in cytosine that attaches to ribose.
iii. Draw a single side chain of arginine such that it forms the most optimally aligned pair of hydrogen
bonds that it can make with the major groove of the G-C base pair. Again, use to
indicate hydrogen bonds. The interaction between arginine and the base pair must not disrupt
base pair hydrogen bonding. Assume that all hydrogens attached to nitrogens in the side chain are
equally effective at forming hydrogen bonds. The structure of the arginine side chain is shown
below.



















5. Briefly explain the primary reason why it is thermodynamically favorable to sequester non-polar amino
acids into the interior of a folded protein.










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6. Two rotated views of the structure of a DNA double helix are shown below. The locations of the water
molecules surrounding the structure are also indicated as dots.

a. Briefly describe how the distribution of water molecules in the structure of DNA speaks to the
thermodynamic driving force that stabilizes the structure of the double helix.












b. Shown below is a structure of a DNA double helix that is bound to the antibiotic netropsin. The
carbons of the DNA molecule are colored pink and the carbons of netropsin are colored in cyan.

In which groove does netropsin bind?



Water molecules
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Netropsin binds this groove in A-T rich sequences. How does the chemical information exposed on
the edge of an A-T base pair differ from the chemical information exposed on the edge of a G-C
base pair in this particular groove?



7. Consider a DNA double helix consisting of dNAM-d5SICS base pairs, as shown on the right. How would
replacing the dNAM-d5SICS base pairs with the altered dNAM- altered d5SICS base pairs shown on the
left affect the stability of the helix (would it become more or less stable)? Briefly explain your answer
in terms of entropy and enthalpy.



























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8. Shown below on the left is an unnatural base pair between isocytosine and isoguanine. To the
right is the natural cytosine:guanine base pair.


a. Label the major groove and minor groove of the unnatural base pair of isocytosine and isoguanine
shown on the left.

b. Label the pattern of hydrogen bond donors and acceptors in the major and minor groove for the
isocytosine:isoguanine base pair.

c. In which groove does a protein interact to determine sequence-specific information about a
segment of DNA? Could a protein distinguish the difference between an isoC:isoG and C:G
base pair? Briefly explain why or why not.


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Answers
1a.
van der Waals interactions (one of the two most important)
the hydrophobic effect (one of the two most important)
hydrogen bonding between Watson-Crick base pairs
b and c.


d. Proflavin, ethidium, and DAPI can bind DNA between base pairs because they share the same flat
aromatic ring structure as the bases so both the hydrophobic effect and van der Waals interactions
will contribute to their favorably base-stacking with the DNA bases.

Moreover they are both large enough because of their multiple rings that they can span the entire
distance of a Watson-Crick base pair. Naphthalene-2,7-diamine is too small: if it intercalated in the
DNA, it would both expose parts of the hydrophobic bases to water and leave van der Waals
interactions with the bases unfulfilled.

2.


3a. The stearic constraints of the double helix make a purine-purine base pair highly unfavorable: there
is not enough space for two purines to fit within the helix without destabilizing the helix, since both
Watson-Crick base pairs (A-T and C-G) are the same width. Moreover, the two purines do not
hydrogen bond donors and acceptors arranged to form the typical Watson-Crick base pair hydrogen
bonds.
b. 8-oxoguanine can base pair with adenine in this orientation because nitrogen 7 is now bound to a
hydrogen rather than having a double bond, allowing it to donate a hydrogen bond to adenine
when the base rotates to relieve stearic stress on the duplex.

Proflavin
Figure A
Figure B
G
G
C
C
3
5
6.8
9
4.




It doesnt matter which nitrogens/hydrogens on arginine are involved in the hydrogen bonds, as
long as they bind to the correct position on the base pair.

5. Exposure of nonpolar sidechains to water causes an ordering of water molecules around the
nonpoloar side chains, which is entropically unfavorable. Sequestering nonpolar side chains in the
interior of a protein prevents ordering of water or releases ordered water, which is entropically very
favorable.

6a. The water molecules are all located along the hydrophilic edges of the bases in the major and minor
grooves or are interacting with the charged and polar sugar phosphate backbone. The waters are
expelled from the hydrophobic interior of the DNA double helix which are occupied by the
hydrophobic bases. The exclusion of water molecules from the central axis of the double helix
where the bases are is a manifestation of the hydrophobic effect, which is the thermodynamic
driving force stabilizing the double helix. If the two complementary DNA strands remained
separated as single strands rather than as a double strand, water molecules would form a solvation
shell around each hydrophobic base, which is very entropically unfavorable.

b. Minor groove. A-T base pairs have the AHA (hydrogen bond acceptor - nonpolar hydrogen
hydrogen bond acceptor) pattern in the minor groove. G-C base pairs have the ADA (hydrogen
bond acceptor hydrogen bond donor hydrogen bond acceptor) pattern in the minor groove.

7. The altered dNAM and d5SICS bases are less hydrophobic and more polar than the original dNAM
and d5SICS bases. Since the primary driving force stabilizing the double helix is the hydrophobic
effect, the more hydrophobic bases will contribute greater stability to the double helix. The more
original, more hydrophobic bases cause more water molecules to adopt an entropically unfavorable
solvation shell when the DNA strands are single-stranded, and therefore more water is released
when the two DNA strands come together to form a double helix. Altering the bases to make them
more polar and less hydrophobic by introducing nitrogens into the original purely carbohydrogen
ring will mean that fewer waters are released upon the formation of the double helix, decreasing
the stability of the double helix if the altered dNAM and d5SICS are included.
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8a and b.

c. Proteins interact with the major groove of DNA base pairs to determine sequence-specific
information about the segment of DNA. Yes, a protein can distinguish isoC:isoG and C:G because
they have different patterns of hydrogen bond donors and acceptors in the major groove (ADA vs
DAA for C:G).

Major
Minor
a a
d
a
a
d