DNA
Why is it important for phosphate ester bonds to have a high activation energy of
hydrolysis?
It is important so that DNA and RNA backbones do not spontaneously fall apart inside cells, despite the fact that hydrolysis would be quite favorable thermodynamically.
The DNA double-helix is intrinsically straight, but flexible. What are two causes of DNA bending?
Transcription factors (binding to proteins)
AT rich sequences
What is DNAs net charge?
Negative
Is DNA gyrase found in human cells?
No
Describe the difference between phosphate esters and phosphoanhydrides, including a (biologically relevant) example.
ATP has both a phosphate ester between its first
phospho group and the 5’-OH of ribose, and two phosphoanhydrides between successive phospho groups. Hydrolysis of either phosphoanhydride releases much more energy than phosphate ester hydrolysis, because it releases charge-charge repulsion between phosphates (and because the products get a particularly happy boost in
resonance stabilization).
A DNA double-helix can be denatured by heating or renatured (possibly) by cooling. Why is complete renaturation trickier than denaturation?
In order for all of the DNA to pair up correctly in renaturation, cooling must happen slowly, so that base-pairs have a chance to form and break again, until the perfect match is made. Otherwise, (if cooling is too fast) the DNA may get “stuck” with noncomplementary parts that cannot pair up.
What are the three types of DNA helix? + Characteristics?
A-DNA:
right handed, 2.6d, 11BP per turn, narrow MG, wide minor G, 19 degree BP tilt
B-DNA
right handed, 2.4d, 10.4 BP per turn, wide MG, narrow minor groove
1 degree BP tilt - bases are largely perpendicular
Z-DNA
left handed, 1.8d, flat MG, narrow minor groove,
9 degree BP tilt
List the chemical and biological differences between DNA and RNA.
RNA - SS molecule
DNA - DS molecule
RNA has a 2' OH which means it degrades faster than DNA which does not have a 2'OH
RNA is used as a temporary messenger, while DNA is used as a permeant "storage" molecule
Explain the shortcomings of the following statement: “Because a G:C base-pair is stabilized by three hydrogen bonds, whereas an A:T base-pair is stabilized by only two hydrogen bonds, GC-rich DNA is harder to melt than AT-rich DNA”
GC rich DNA is harder to "melt" than AT rich DNA, however, this is NOT due to the greater quantity of hydrogen bonds.
The bases could hydrogen bond with water in the same fashion if separated, therefore, these hydrogen bonds aren't what makes the GC base pair stronger.
The right answer is that GC base pairs have stronger pie stacking than AT base pairs
What provides the main driving force favoring double-helix formation? How are hydrogen bonds involved in double-helix formation? Why is it unlikely to see two purines base-pair with each other in a double-helix?
Enthalpy release by Pi stacking
Hydrogen bonding allows the base pairs to pair up in a regular way that allows pi stacking to occur
Two purine bases would not fit properly, and would create a bulge in the DNA double helix + they would most likely NOT have compatible H bonding pairs
Relaxed DNA has how many turns per BP
10.4
Ciprofloxacin inhibits DNA gyrase. Why is Cipro a relatively safe antibiotic? Compare and contrast DNA gyrase and topoisomerase I.
Cipro is a relatively safe antibiotic because it targets an enzyme that is not present in human cells (DNA gyrase)
DNA gryase cuts both DNA strands (of one duplex) while topoisomerase I only cuts one of the strands.
Topoisomerase I only allows for 1 "turn" / supercoil to be released while DNA gryase allows 2
Both reseal the strands afterwards
DNA gryase can create negative supercoils, while topiosomerase I can only relax DNA
What is cooperativity? How does cooperativity apply to DNA?
Cooperativity is when multiple things have a weaker effect individually than they do together.
In DNA, the pie stacking interactions are relatively weak on their own (there are just london dispersion forces), however, as a collective group (all the BPs in DNA) these pie stacking reactions are very strong and favorable. Cooperativity is what makes pie stacking strong AND what leads to the favorable formation of the DNA double helix structure.
List two similarities and four differences between positive supercoiling and negative supercoiling.
similar: both are changes away from the “relaxed” most energy-favored amount that DNA strands twist around each other, both can cause superhelical writhes to form.
differences: negative supercoils are most commonly found in vivo, have fewer turns than relaxed DNA and make it easier to pull the DNA strands apart; positive supercoils have more turns than relaxed DNA and make it harder to pull the DNA strands apart; more positive supercoils accumulate in the remaining helix when strands do get pulled apart.
Why does DNA usually prefer to form a B-helix? Why does mRNA not form a B-helix? What does mRNA do instead? RNA-DNA hybrids are possible, where one strand of the double helix is RNA and the other is DNA. What form would you expect such a helix to take? Explain.
DNA usually prefers to form a B-helix since it offers the strongest pi stacking interactions
mRNA does NOT form a B-helix due to steric hinderance
mRNA is a SS molecule, although it can sometimes base pair with itself and form hair pin loops
RNA-DNA hybrids would form an A-helix
Compare and contrast human type I and type II topoisomerase.
Human type I topiosomerase cleaves only one strand of the DNA double helix while type II cleaves both strands
Both are only capable of relaxing DNA NOT inducing negative supercoiling.
Type I removes one supercoil while type II removes two.
Both reseal the DNA after removing the supercoil (s)
Explain the molecular events of denaturation and renaturation of DNA. Describe how these terms apply to PCR.
In DNA denaturation heat/energy must be added to allow the pie stacking interactions to "break".
DNA renaturation, however, is a spontaneous process since the pie stacking bonds that hold the DNA double helix together are very favorable.
In PCR we use heat to denature DNA, and then we use a temperature that is just cold enough to allow the strands to renature, but not so cold that they renature improperly.
A CG base-pair has additional H-bond donors/acceptors in the major groove, matching the pattern donor-acceptor-acceptor. What does this mean? How does this affect DNA’s interactions with water? How does this affect DNA’s possible interactions with a sequence-specific DNA-binding protein?
This distinctive pattern is exposed on the edge of each CG base-pair, on the surface that
faces into the major groove.
Water molecules in the major groove will H-bond (favorable dipole-dipole interactions) with those exposed donors/acceptors.
A sequence-specific DNA binding protein could fit snugly into the major groove only when H-bonding partners on the protein match up with the ones exposed on the edge of its target DNA base-pairs, to replace the H-bonds that would otherwise be made with water.
Compare and contrast positive and negative supercoiling. Which type of supercoiling is most commonly found in vivo? Which type of supercoiling can be caused by separating strands and opening a “bubble” in the double helix? What are two reasons why supercoiling is useful in vivo?
Positive supercoiling results in more "turns" in the DNA while negative supercoiling results in fewer "turns."
Both require energy, and are steps away from the most favorable / lowest energy form of DNA
Negatively supercoiled DNA is most common in vivo
Positive supercoiling can be formed at the ends of a DNA bubble
Negative supercoiling can be used in vivo to make the DNA strands easier to pull apart, therefore, making processes such as DNA replication and transcription easier
(Negative supercoiling occurs when DNA is wrapped around histones)
Positive supercoiling can also be used in vivo to "silence" sections of DNA / make them harder to pull apart.
What does topoisomerase I do? How does it do it? What is the difference between topo I and an enzyme that simply produces single-stranded nicks in DNA (a “nickase”)?
Topo I nicks one strand of the DNA backbone (holding onto the ends), passes the other strand through the gap (once), then reseals the nick, resulting on one less supercoil.
Difference between a nickase:
Passes the other DNA strand through the gap
Reseals the strand