Lecture 2: DNA Polymerases and Chemistry of DNA Synthesis

Question 1

What were the three suggested models of DNA Replication until the 1950’s? Explain each model

Answer 1

• Conservative Replication: Parental DNA stays together, and the newly formed daughter strands stay together
• Semi-conservative Method: Two of the parental DNA strands act as a template for the new DNA to be synthesized (copied). After replication, each double stranded DNA helix has one copy of the parental DNA strand and one new daughter strand
• Dispersive Model: both copies of the DNA have segments of the parental DNA strand and the new DNA stranded mixed together.

Question 2

Which experiment provided the semi-conservative method to be true?

Answer 2

Meselson-Stahl experiment in which cells were grown for many generations in mediums containing various isotopes of nitrogen.
- First medium had original heavy DNA of N15
- Second medium had generation 1 with hybrid DNA of N15 and N14
- Third medium had generation 2 with light DNA (N14) and hybrid DNA (N15 and N14)
This confirmed semi-conservative replication as continuation replication of the molecules produced DNA with 1 parental DNA strand and one replication strand.

Question 3

Name the 4 nucleotides of DNA, classify each as either a purine or pyrimidine.

Answer 3

Pyrimidine: Cytosine and Thymine
Purine: Adenine and Guanine

Question 4

What are the key substrates required for DNA synthesis?

Answer 4

1. Four deoxynucleoside triphosphate (dNTPs) –> dATP, dTTP, dCTP, dGTP
2. Primer:template junction

Question 5

Describe the function of the primer template junction in DNA synthesis.

Answer 5

This template provides the single stranded DNA (ssDNA) which will help direct the addition of each complementary deoxynucleotide. The primer is also complementary to the template and it must have an 3’OH adjacent to the single strand region as this is where the nucleotide will be added.

Question 6

What is the fundamental reaction that occurs in DNA synthesis? How does it occur?

Answer 6

The reaction is a phosphoryl group transfer. The 3’OH group of the nucleotide at the end of the growing strand acts as a nucleophile. Nucleophilic attack can then occur at the 5’ phosphate of the incoming dNTP. Inorganic pyrophosphate is released due to this reaction.

Question 7

What is the change in free energy for a DNA synthesis reaction?

Answer 7

There is a minimal change in free energy because the phosphodiester bond that is formed

Question 7

What is the change in free energy for a DNA synthesis reaction?

Answer 7

There is a minimal change in free energy because the phosphodiester bond that is formed at the expense of a less stable phosphate anhydride and there is not a large difference between the bonds.

Question 8

What role does the enzyme pyrophosphatase play in DNA synthesis?

Answer 8

Converts the released pyrophosphate molecule into two phosphates which helps to make the forward reaction of nucleotide addition to be the more favourable reaction.

Question 9

What are the 3 main properties of DNA polymerase?

Answer 9

1. Catalytic flexibility
2. Kinetic proof reading
3. Substrate specificity

Question 10

Describe catalytic flexibility as a property of DNA polymerase.

Answer 10

DNA polymerases only use a single active site in order to catalyze the addition of any of the four dNTPs at the primer: template junction. This flexibility is due to the nearly identical geometry of the A:T and G:C base pairs.

Question 11

Describe kinetic proofreading as a property of DNA polymerase

Answer 11

DNA polymerase is able to kinetically proof read the addition of incoming dNTPs because unfavourable alignment of the substrate (aka, wrong nucleotide being added) will lead to dramatically lower rates of nucleotide addition (10 thousand fold slower).

Question 12

Describe substrate specificity as a property of DNA polymerase.

Answer 12

DNA polymerases are able to distinguish between rNTPs and dNTPs very well. Cells have 10 fold higher concentration of rNTPs in them, but they are incorporated into the DNA at a rate of 1000 fold lower than dNTPs. This is due to steric exclusion of the incoming dNTP.

Question 13

What is the role of Magnesium ions in the catalytic mechanism of DNA polymerase?

Answer 13

Magnesium ions help to form phosphate groups with incoming dNTPs. This occurs because two Magnesium ions (Mg 2+) are bonded to the phosphate groups of the incoming dNTP and to three Asp residues in its active side. This helps to facilitate the nucleophilic attack of the 3 hydroxyl group of the primer on the phosphate of the dNTP and facilitate the displacement of the pyrophosphate.

Question 14

Name the three parts of the primer: template junction as if they represent a hand.

Answer 14

The palm domain, the thumb domain, and the fingers domain

Question 15

What is the function of the palm domain in the primer:template junction?

Answer 15

This domain monitors the base pairing of the recently added nucleotides. There are two metal ions (Mg2+ and Zn2+) at the catalytic center which are essential for the activity of the primer:template junction.

Question 16

What is the function of the thumb domain in the primer:template junction?

Answer 16

This domain interacts with the most recently synthesized DNA. This helps to maintain the correct position of the primer and the active site. It also helps to maintain strong association between there DNA polymerase and its substrate which helps the DNA polymerase add many dNTPs at one time when it binds to the primer:template junction.

Question 17

What is the function of the fingers domain in the primer:template junction?

Answer 17

This domain helps to move the incoming dNTP into close contact with the active site. This is because once the correct basepair has been added, the finger domain will move to enclose the dNTP. This helps stimulate catalysis because it moves the incoming nucleotide into close contact with the catalytic metal ions.

Question 18

What is the catalytic speed of DNA polymerase and why is it possible?

Answer 18

1000 nucleotides / second and it is possible due to its processivity

Question 19

What does processivity mean?

Answer 19

Refers to an enzymes ability to catalyze consecutive reaction without releasing its substrate. In the case of DNA polymerase, the degree of processivity is defined as the average number of nucleotides that are added each time the enzyme (DNA Pol) binds to the primer:template junction.

Question 20

Is initial binding of the polymerase to the primer:template junction the rate limiting step for DNA synthesis.

Answer 20

Yes, once it is bound, the rate of DNA synthesis is very high

Question 21

What are some contributing factors to the accuracy of cellular DNA synthesis?

Answer 21

Base pair geometry and complementarity between the bases

Question 22

What accounts for the high degree of precision in replication and low mutation rate that is seen in physiology conditions?

Answer 22

Proof reading exonuclease activity of DNA polymerase

Question 23

How does proof reading exonuclease activity work in DNA polymerase?

Answer 23

Proof reading exonuclease help to double check and remove any incorrect nucleotides that are added at the 3’ end allowing the DNA polymerase a second chance to add the correct nucleotide. Since DNA synthesis happens in the 5’ to 3’ direction, the exonuclease works in the 3’ to 5’ direction to ensure the DNA pol can be given that second chance.

Question 24

How does base pair geometry also help the proofreading exonuclease activity?

Answer 24

Altered geometry due mis paired DNA bases slows down the DNA polymerase which gives the exonuclease more time to work in removing the incorrect bases and to double check.

Question 25

What is the accuracy of replication due to the help of the proof reading activity?

Answer 25

~1 mistake in every 10^7 nucleotides

Question 26

What are the three types of DNA polymerases in E.coli?

Answer 26

DNA polymerase I, DNA polymerase II, and DNA polymerase III

Question 27

Describe DNA polymerase II of E.coli

Answer 27

Is an enzyme that is involved in one type of DNA repair.

Question 28

Describe DNA polymerase III of E.coli

Answer 28

Principal replication enzyme in E.coli. It is considered the most important as it responsible for major replication processes.`

Question 29

Describe DNA polymerase I in E.coli

Answer 29

Performs many clean up functions during replication (removal of primers), during recombination, and during repair (nick translation). The special functions of DNA polymerase I are enhanced by its 5’ to 3’ exonuclease activity (not used for proof-reading like the 3’ to 5’ exonuclease but rather for removal of primers + in repair pathways)

Question 30

What happens when the 5’ to 3’ exonuclease domain is removed from DNA polymerase I?

Answer 30

The remaining fragment, known as the Klenow fragment or the large fragment, retains the polymerization and proof reading activities.