DNA Replication

Question 1

Explain the idea of DNA replication being semiconservative.

Answer 1

This proposes that during replication, the 2 original DNA strands separates, with each serving as a template strand to a new DNA strand to be synthesised. Thus, the resulting 2 DNA strands are made up of half the template strand and half the new strand.

Question 2

How do we know that DNA replication is semiconservative?

Answer 2

Meselson and Stahl conducted an experiment. They cultured bacteria in a 15N medium, then shifted the bacteria to a 14N medium, DNA was isolated at different times corresponding to replication cycles 0, 1, and 2.

After 1 replication cycle, the DNA was all intermediate density. This rules out the conservative theory.

After 2 replication cycles, 2 bands of DNA were seen, with half being intermediate and half being light. This proves the semiconservative theory.

Question 3

What is a replication origin?

Answer 3

Particular sequence of DNA at which replication is initiated.

Question 4

Which directions does the replication forks move?

Answer 4

Bilaterally

Question 5

What is the function of DNA polymerase?

Answer 5

DNA polymerase caltalyzes the addition of nucleotides to the 3’ growing end of the DNA strand.

Question 6

Why is there a leading and lagging strand?

Answer 6

Because DNA polymerase can only add nucleotides to the 3’ growing end, therefore they can only move in the 5’-3’ direction.

Consequently polymerase moves backwards on the other strand, synthesizing small, discontinious DNA pieces.

This strand of DNA lags behind the other one will the polymerase can move continiously.

Question 7

Describe the structure of DNA polymerase and how this helps it carry out its function.

Answer 7

DNA polymerase has a polymerizing (P) and editing (E) mode.

If the wrong nucleotide is added, the newly synthesised DNA strand unpairs from the template DNA strand and its 3’ end moves from P site to E site to be removed.

Question 8

Why are okazaki fragments formed on the lagging strand?

Answer 8

Because the DNA polymerase can only catalyse the addition of nucleotides in the 5’-3’ direction, on the lagging strand, it synthesises DNA in small, discontinious pieces.

These small pieces of DNA form the okazaki fragments.

Question 9

Describe the function of primase.

Answer 9

Primase synthesises RNA primers.

RNA primers are needed as they are a short length of RNA that can base-pair with the template strand to provide a base-paired 3’ starting end for DNA polymerase.

On the leading strand, a RNA primer is only needed at the replication origin. Though on the lagging strand, new primers are continually laid down at intervals along the exposed DNA template strand, in order to keep polymerization going.

Consequently, primers make many mistakes.

Question 10

Describe the function of DNA helicase.

Answer 10

DNA helicase lies at the front of the replication machine to open up DNA ahead of the replication fork.

It uses ATP to propel itself foward.

Question 11

How does DNA polymerase stay on the DNA strand?

Answer 11

DNA polymerase needs a sliding clamp to stay on the DNA strand, this is in turn loaded by the clamp loader.

The clamp loader loads the sliding clamp onto the DNA. ATP binds to the clamp loader, which opens the sliding clamp. Then DNA is secured inside the clamp.

After that ATP hydrolysis, locking the sliding clamp around the DNA and releasing the clamp loader. Consequently DNA polymerase is bound to the DNA.

This is only done once on the leading strand, but many times on the lagging strand.

Question 12

Why can DNA tangle during replication?

Answer 12

Because the force of the replication fork causes tension to build up in DNA, which can cause them to tangle.

Question 13

What is the mismatch repair system?

Answer 13

A system that removes errors not detected by the replication machine. These errors are removed after replication.

Question 14

How does the mismatch repair system work?

Answer 14

There are 2 proteins involved - Mut S (scanner) and Mut L (enzyme).

When Mut S detects an error, a loop is produced in that area, which is larger than the error itself. Then Mut L cuts the new strand and degrades the whole region.

DNA synthesis is repaired using the template strand.

Question 15

How do single strand proteins prevent DNA tangling during replication?

Answer 15

They cling to the single strand of the double helix exposed by helicase, thus keeping them elongated so they can be efficient templates.

Question 16

How does initiator proteins begin the replication process?

Answer 16

Initiator proteins bind to replication origins to destabilize the AT-rich sequences. This can start the replication process.

Question 17

How does prokaryotic cells ensure they have sufficient nutrients for replication?

Answer 17

If there is enough nutrients for a round of replication, initiation occurs. The new DNA strand is not immediately methylated while the template ones are, resulting in as state of hemimethylation. This impairs replication, therefore the cell will check the nutrient condition before a new replication round begins.

Question 18

Why are telomere needed?

Answer 18

The replication fork can’t replicate all the way to the end of the DNA strand in the lagging strand, due to the backstitching method.

Therefore telomeres are needed to complete the replication process.

Question 19

How does telomeres function?

Answer 19

Telomeres are long, repetitive nucleotide sequences which attractes telomerase. They have their own RNA template to extend the end of the template lagging strand. Then replication continious as normal.

This also forms structures that mark the true end of a DNA, allowing the cell to distinguish a natural end vs a double strand break.

Question 20

How does DNA polymerase catalyze the addition of a nucleotide?

Answer 20

The polymerization reaction involves a phosphodiester bond between the 3’ end of the growing strand and 5’ phosphate group of the new nucleotide.

This enters the reaction as deoxyribonucleoside triphosphate, one of its high energy phosphate bond is hydrolysed to provide energy for the linking of the nucleotide to the chain.

This released pyrophosphate, which is again hydrolyzed into inorganic phosphate, therby making the polymerization reaction irreversible.

Question 21

How does DNA polymerase reduce the chances of mistakes?

Answer 21

DNA polymerase carefully monitors the incoming nucleotide and the template strand for correct base pairing.

Though if incorrect, it corrects the error through proofreading. This means that before adding the next nucleotide, polymerase checks the previous ensure it’s correct. If not, polymerase will clip off the nucleotide to try again.

This is carried out by a nuclease that cleaves the phosphodiester backbone.

Question 22

How is the replication process completed on the lagging strand?

Answer 22

A nuclease degrades the RNA primers (RNAse H), a DNA polymerase called repair polymerase then replaces this with DNA using the the end of the adjacent okazaki fragments as primers. Lastly DNA ligase joins the 5’ phosphyl end to the adjacent 3’ hydroxyl end.