DNA replication

Question 1

Learning Objectives

Answer 1

• How the DNA replication machinery assembles at the replication fork
  • Describe Leading and lagging strand DNA synthesis
  • Role of DNA helicases, DNA polymerases and topoisomerase in DNA replication
  • How DNA synthesis is regulated by cell cycle control proteins
  • Describe replication finishing of linear chromosomes
• Role of telomeres and telomerase and the consequences of disease when something goes wrong.

Question 2

Describe Leading and lagging strand DNA synthesis

Answer 2

Leading Strand DNA Synthesis:

1 - Continuous Synthesis:

The leading strand is synthesized continuously in the 5’ to 3’ direction, matching the direction of the replication fork movement.
DNA polymerase adds nucleotides in the 3’ to 5’ direction, synthesizing the new strand in the same direction as the replication fork advances.

2- Single Primer:

The leading strand requires a single RNA primer to initiate DNA synthesis.
The primer is complementary to the template strand and serves as the starting point for DNA polymerase.

3- High Processivity:

DNA polymerase has high processivity on the leading strand, meaning it can continuously add nucleotides without frequently detaching from the template.

4- Faster Replication:

The leading strand is replicated at a faster rate than the lagging strand due to its continuous synthesis.

Lagging Strand DNA Synthesis:

Discontinuous Synthesis:

The lagging strand is synthesized discontinuously in short fragments known as Okazaki fragments.
DNA polymerase synthesizes each Okazaki fragment separately in the 5’ to 3’ direction, opposite to the movement of the replication fork.

Multiple Primers:

The lagging strand requires multiple RNA primers, each serving as the starting point for the synthesis of an Okazaki fragment.

Okazaki Fragment Processing:

After the synthesis of Okazaki fragments, RNA primers are replaced with DNA by DNA polymerase and DNA ligase.
This process results in a continuous lagging strand.

Lower Processivity:

DNA polymerase on the lagging strand has lower processivity compared to the leading strand, as it frequently detaches after synthesizing each Okazaki fragment.

Question 3

Role of DNA helicases, DNA polymerases and topoisomerase in DNA replication

Answer 3

DNA Helicases:

Function:
DNA helicases are enzymes responsible for unwinding the double-stranded DNA at the replication fork.
DNA helixases break the hydrogen bonds between complementary nucleotides, separating the DNA strands and creating a single-stranded template for replication.

Role in DNA Replication:
DNA helicases work ahead of the replication fork, facilitating the movement of the DNA polymerase along the template strand.
Their unwinding activity is essential for exposing the template DNA and allowing the synthesis of new complementary strands.

DNA Polymerases:

Function:
DNA polymerases are enzymes that catalyze the synthesis of new DNA strands by adding complementary nucleotides to the template strand.
They require a primer (either RNA or DNA) to initiate synthesis and elongate the growing DNA chain in the 5’ to 3’ direction.

Role in DNA Replication:
DNA polymerases are central to the process of DNA replication, ensuring accurate and faithful duplication of the genetic material.
They synthesize the leading and lagging strands, working in coordination to replicate the entire DNA molecule.

Topoisomerases:

Function:
Topoisomerases are enzymes that regulate the winding and unwinding of DNA strands, preventing excessive coiling or tangling.
They relieve the torsional stress that builds up ahead of the replication fork during unwinding by helicases.

Role in DNA Replication:
Topoisomerases play a critical role in maintaining the structural integrity of the DNA during replication.
Topoisomerases cleave one or both strands of the DNA, allowing it to rotate and release the built-up tension before resealing the break.
Topoisomerases ensure the smooth progression of the replication fork by preventing supercoiling.