Lecture 1 Notes: MITx: 7.28.

1x Molecular Biology: DNA Replication and Repair

Week 1

GOALS:
1. Understand how DNA polymerase catalyzes the addition of deoxynucleoside
triphosphates (dNTPs) to a growing DNA strand.
2. Learn experimental assays for detecting and characterizing DNA polymerase
activity.

OBJECTIVES:
After completing the materials for this week, you should be able to...
1. Recognize the structure of nucleic acids and key features of the structure of
DNA polymerase.
2. Describe the factors that confer high accuracy to DNA polymerase activity.
3. Given the function of a DNA-binding protein, predict if the protein binds the
major or minor groove of DNA.
4. Summarize how critical residues of DNA polymerase and metal ions facilitate
catalysis.
5. Illustrate how a tautomeric state will result in addition of the wrong base.
6. Design the best experiment to test DNA polymerase activity given your
hypothesis that you want to test and experimental constraints.
7. Define processivity in both general terms and specifically in regards to DNA
polymerase.
8. Troubleshoot negative results from a hypothetical DNA polymerase processivity
assay.
9. Interpret data from DNA replication experiments.
10. Analyze protein structures to infer functional information.

Segment 2:
1. The most common form of the DNA double helix is called B-form DNA
2. Each strand of DNA is a polynucleotide meaning the strand is made up of many individual units
called nucleotides

3. A nucleotide has three components: a five carbon sugar, a phosphate group, and one of four
possible nitrogenous bases (adenine, guanine, thymine, and cytosine)

4. The nitrogenous base is always attached at the 1' carbon of the sugar and there is a phosphate
between the 5' carbon of one sugar and the 3' carbon of the neighboring sugar

5. The sugar is deoxyribose and lacks a hydroxyl group at the 2' carbon which is present in ribose
6. Nucleotides in the DNA strand are attached by phosphodiester bonds where the phosphate
group of one nucleotide binds to the 3' oxygen of the neighboring nucleotides

7. Sugars and phosphate groups make up the DNA backbone. The carbon numbering is key to
describing the directionality of the DNA strand, 5' to 3'. In the image below the top strand is
shown 5' to 3' and the bottom strand is 3' to 5' (aka, respectively Watson and Crick).

8. The DNA backbone of deoxyribose and phosphates is bound by covalent bonds whereas the two
DNA strands interact through non-covalent hydrogen bonds between the bases. Thymine
preferentially pairs with adenine through two hydrogen bonds and cytosine preferentially pairs
with guanine through three hydrogen bonds. T and C are known as pyrimidines characterized by
their single ring structure whereas A and G are purines with double ring structures.

9. The geometry of the AT and GC base pairs is the same which allows for symmetry and base
stacking whereas other base pairs cannot form strong hydrogen bonds and do not have the same
geometry
10. The double helix structure of DNA is highly regular, each turn of the helix measures
approximately ten base pairs.
11. Pi-pi interaction form when aromatic rings of bases stack next to each other resulting in base
stacking which stabilizes the double helix structure
12. The double helix forms two repeating structures called major and minor grooves which act as
base pair recognition and binding sites for proteins. The major groove contains base pair
specific information while the minor groove is largely base pair nonspecific due to the patterns
of hydrogen bond acceptors and donors that proteins can interact with in the grooves.

Segment 3:
1. DNA Replication most important reason is to replicate cell's genome
2. Consequences of errors in replication are more often bad than good so accuracy is important
3. DNA replication
1. Rapid: 100 1,000 bp/sec
2. Complete: essentially every base pair must be replicated in the genome
3. Accurate: 1 mistake in every 10,000,000,000 bp synthesized
Segment 4:

1. Substrate for DNA replication (primer template junction)

2. The primer has a free 3' OH (hydroxyl) group which attacks the alpha phosphate of an incoming
deoxynucleotide triphosphate, releasing a pyrophosphate. This only occurs when this base
correctly base pairs with the base on the template.

3. This reaction progresses because the cleaving of the resulting pyrophosphate (two phosphates
coupled to one another) has a G = -7 kcal, whereas the reaction without would have G = -3.5
kcal. This makes the reaction irreversible.
4. DNA Polymerase catalyzes DNA synthesis and requires the following:
1. A 3' OH primer
2. Annealed to a longer ss DNA (w/ ss DNA adjacent to the 3' OH primer)
3. Items 1 & 2 above constitute a Primer Template Junction (PTJ)
4. All 4 dNTP's (dATP, dGtp, dCTP and dCTP)
5. Added dNTP must base pair with the template for catalysis
6. 3' OH primer is extended by DNA synthesis ( often referred to as 5' to 3' )

Segment 5:
1. How many reactions does DNA Polymerase catalyze?
1. Four, one for each base plus four more considering the neighboring base pair as part of the
reaction for a total of 16 reactions (There are potentially many more reactions if one
considers bases further upstream of the active site).
2. It catalyzes these reaction using a single active site

2. How does one active site catalyze the addition of multiple dNTPs? The structure of DNA is
very regular which makes it possible for the DNA polymerase to add bases with no concern for
which particular base is being added. As shown below the bases have very similar structures
(with minor differences) and nearly identical dimensions so that the resulting DNA has a very
regular structure with consistent dimensions throughout as shown below.

3. Correct base pairing places phosphate group in correct position for SN2 nucleophilic attack
by the 3' OH of the adjacent base which causes release of pyrophophate from the dNTP.

4. Mismatched bases bind so slowly that the mispaired nucleotide usually floats out of the active
site before the reaction can complete
5. There are both temporal and affinity mechanisms to ensure correct base pairing
Segment 6:
1. What does DNA polymerase look like? The fingers move while the thumb is stationary.

2. DNA polymerase structure:

1. Palm binds PTJ and newly synthesized DNA

2. DNA polymerase palm binds all DNA sequences by both interacting with the phosphate
backbone and the minor groove since the pattern of hydrogen bond acceptors is the same for
every base pair.

3. There are interactions that are non-sequence specific but Watson/Crick base pair specific in
the minor groove. Many proteins that recognize DNA non-specifically use these interactions
to do so.
4. Non-base pairs have different hydrogen bond acceptor patterns and can be recognized by
these different patterns.

Segment 7:
5. dNTP binding is mediated by base pairing and binding to divalent metal cation (often Mg+2)

6. Finger domain closes on bound dNTP and prevents hydrolysis from occurring allowing
hydroxyl group to attack the alpha phosphate
1. O-helix ( helix)
2. Tyr forms pi-pi bonds with base
3. Lys and Arg bind beta and gamma phosphates of triphosphate

7. Release of the pyrophosphate releases the O-helix

8. Primer slides one bp down in polymerase to reposition OH for next dNTP addition

Segment 8:
1. DNA polymerase makes one mistake per 105 bp synthesized
2. Most mistakes are pyrimidine pyrimidine (C T) or purine purine (G A)
1. Due to tautomer formation

2. Tautomer formation changes hydrogen bonding patterns which changes base pairing
preference

3. Once paired, however, the nucleotides rapidly revert to their original state which causes the
end of the newly synthesized DNA strand to fray

4. This then moves the 3' OH out of position to interact with the alpha phosphate which slows
synthesis and provides time for repair to take place using a proofreading exonulease. So
tautomeric mispairing is temporary.

Segment 9:
1. proofreading exonucleases remove mismatches
1.
2.
3.
4.
5.

exonuclease degrades DNA from one end

Proofreading exonuclease acts in 3' 5' direction
Typically part of the same polypeptide as the polymerase
Frayed/non-bp end of DNA has low affinity for polymerase active site
ssDNA end of primer 3' OH end has high affinity for exonuclease active site

2. Exonuclease process:
1. Mismatch is poor substrate for polymerase

2. causing single stranded region to come out of active site and into exonuclease active site

3. where exonulease removes one to three nucleotides

4. DNA strands then reanneal and shift into position of polymerase active site
3. Proofreading exonucleases increase accuracy to one mistake in 107 bp synthesized