Doubling the Double Helix: DNA Replication Quiz

Quiz Answer Key and Fun Facts

1. The basic mechanism of DNA replication was determined by the Meselson-Stahl experiment. In this experiment, they bred bacteria in nitrogen-15 (N-15) media for many generations, which had the effect of making the bacterial DNA denser than normal DNA (which consists mostly of nitrogen-14). They then allowed the bacteria to reproduce in nitrogen-14 (N-14) media for a set number of generations. When they isolated the bacterial DNA from bacteria allowed to reproduce for two generations in the N-14 media and suspended the DNA in a solution with a density gradient from top to bottom (meaning denser materials tend toward the bottom and less dense materials tend toward the top), they saw two distinct bands of DNA at different density levels. Even without any additional data, which mode of DNA replication was immediately ruled out?

Answer: dispersive

In dispersive replication, you would almost never see multiple distinct bands (because all the DNA strands would have roughly equal ratios of the old N-15 DNA and the new N-14 DNA and be identical in density). It turns out that of the two bands of DNA they saw, one band was roughly as dense as plain N-14 DNA and the other was halfway in between the density of N-14 and N-15 DNA (if you aren't convinced, draw some diagrams and see how semi-conservative replication carried out twice starting with a single 100% N-15 DNA strand leads to two pure N-14 DNA strands and 2 mixed N-14/N-15 DNA strands; remember that all the new strands of DNA are made using N-14).

This additional information heavily favors the semi-conservative mode of replication, and the data from subsequent generations of bacteria pointed to the same conclusion.

2. DNA replication starts with enzymes called helicases separating the DNA double helix into two separate strands, a process that requires energy. Is it true that helicases can only get this energy from ATP hydrolysis?

Answer: No

Helicases may derive energy from ATP or GTP hydrolysis. After helicase separates the two strands of a double helix, single-stranded binding proteins (SSBP) attach to the separated strands of DNA to prevent them from reattaching to each other, since the double-stranded helix is the thermodynamically favored state.

3. The enzyme that does most of the work in DNA replication is DNA polymerase III. Besides DNA, what are this enzyme's substrates?

Answer: deoxynucleoside triphosphates

It's *DNA* polymerase, so obviously it's going to want to work with deoxynucleosides (nucleosides with deoxyribose instead of ribose). Although the nucleotides found in DNA are deoxynucleoside monophosphates, DNA polymerase III requires deoxynucleoside triphosphates (dNTPs). Why? Well, answer the next question to find out...

4. What type of chemical reaction does DNA polymerase III catalyze when it synthesizes new DNA?

Answer: nucleophilic substitution

DNA polymerase III catalyzes a nucleophilic attack by the 3' OH group of the deoxyribose on the innermost phosphate group of a dNTP. This causes a pyrophosphate ion to be released (two phosphates covalently linked). The energy released from the hydrolysis of the pyrophosphate into 2 orthophosphate ions is used to drive the DNA synthesis reaction forward.

Hence, this is why the polymerase works with dNTPs instead of dNMPs.

5. Besides a nucleotide primer with a 3' OH and a dNTP, what else does DNA polymerase III need to do its job?

Answer: metal ion

DNA polymerase uses two metal cations (usually Mg2+). One interacts with both the primer OH and the dNTP, coordinating the nucleophilic attack. The other interacts only with the dNTP, and helps to stabilize the negative charge on the pyrophosphate that is produced by the reaction.

6. DNA polymerases have high fidelity because they can easily tell the difference between an A-T pair and a G-C pair because A-T pairs have hydrogen bond acceptors only on the minor groove side while G-C pairs have hydrogen bond acceptors only on the major groove side.

Answer: False

Both A-T and G-C pairs have hydrogen bond acceptors on the minor groove side. DNA polymerases donate two hydrogen bonds to these acceptors. The high fidelity of DNA polymerases mainly come from the fact that when a dNTP enters the active site of the polymerase, it triggers a conformational change in the enzyme that forms a tight pocket that will only readily fit properly shaped base pairs inside.

7. In synthesizing the LAGGING STRAND of DNA, which of the following enzymes are needed? I. DNA Polymerase III II. DNA Polymerase I III. DNA Ligase IV. Topoisomerase II

Answer: I, II, III, and IV

ALL of the enzymes listed are needed. DNA polymerase III does most of the work by catalyzing the addition of new nucleotides to the strand. Topoisomerase II works to relieve torsional stress on the DNA molecule caused by the unwinding at the replication fork. DNA Polymerase I is needed to remove the RNA primers from the Okazaki fragments with its 5'-3' exonuclease activity. Finally, DNA
ligase is needed to join the Okazaki fragments together with phosphodiester linkages.

8. In bacteria, NAD+ is used as the energy source for DNA ligase.

Answer: True

DNA ligase in eukaryotes and archea hydrolyzes ATP to AMP and pyrophosphate while in bacteria it is generally NAD+ that gets cleaved to form AMP and nicotinamide mononucleotide (NMN).

9. Both DNA polymerase III and DNA polymerase I have the so-called 3'-5' exonuclease activity. What is this activity used for?

Answer: proofreading

Earlier it was mentioned that DNA polymerases have a high fidelity due to their strict discrimination between correct and incorrect base pairing during 5'-3' synthesis. However, their fidelity is also enhanced by their ability to proofread, or double-check the base pairing of the last nucleotide pair that was synthesized. If the pairing is incorrect, the polymerase is able to remove that nucleotide (3'-5' exonuclease means it can only remove a nucleotide from the end of a DNA strand in the 3' to 5' direction, hence why these polymerases can only proofread the last nucleotide pair they synthesized). Once the nucleotide is removed, 5'-3' synthesis continues again to re-synthesize the (now-removed) incorrect nucleotide.

10. A problem with lagging strand DNA synthesis in linear DNA is that synthesis cannot continue beyond the last RNA primer once the replication fork nears the end of the parental DNA strand. The effect of this is that essentially every time linear DNA replicates, a small section of both ends are cut off. Eukaryotes have developed a solution to this problem by adding what to the ends of chromosomes?

Answer: telomere

Telomeres are essentially filler at the ends of eukaryotic chromosomes. They are sections of DNA that contain no genes or any other useful sequence information. They are there to ensure that when the ends of chromosomes are cut off during replication, it's filler that gets cut off and not sequences that may be useful. Telomeres have other functions, such as protecting the ends of the chromosomes. Enzymes called telomerases have the ability to elongate telomeres, though in most cells these enzymes are not functional once the cell reaches a certain age.

It has been hypothesized that telomere shortening may be a factor in the aging process, since in adult organisms telomeres cannot normally be elongated. Therefore, after an organism's cells have divided and their chromosomes replicated enough times, the telomeres on those chromosomes will eventually disappear, and further replication will then destroy useful genetic information at the ends of chromosomes.

Source: Author aznricepuff

This quiz was reviewed by FunTrivia editor crisw before going online.
Any errors found in FunTrivia content are routinely corrected through our feedback system.

My second DNA quiz...this time it's on DNA replication.

NEXT>

NEXT>

NEXT>

NEXT>

NEXT>

NEXT>

NEXT>

NEXT>

NEXT>