Replisome Assembly

Question 1

What is the replisome?

Answer 1

The massive complex of proteins assembeled at each replication fork.

Question 2

What does the typical replisome at one replication fork consist of?

Answer 2

2 DNA polymerase III molecules (1 each for leading and lagging strands), 1 DnaG, 1 DnaB

Question 3

How many replisomes does a single replication bubble have?

Answer 3

2; 1/fork

Question 4

What are the three subunits of DNA polymerase III?

Answer 4

alpha, epsilon, theta

Question 5

What is the function of the alpha subunit in DNA pol III?

Answer 5

5’ to 3’ polymerase, site of major catalytic function

Question 6

What is the function of the epsilon subunit in DNA pol III?

Answer 6

Removes nucleotides in 3’ to 5’ direction, used for proofreading.

Question 7

What is the function of the theta subunit in DNA pol III?

Answer 7

Stimulates epsilon subunit

Question 8

What does the beta clamp do?

Answer 8

Binds to ssDNA, DNA pol III uses it as the foundation to bind to DNA.

Question 9

What is the function of the clamp loader-TauC complex?

Answer 9

Opens beta clamp (which has a donut shape) so that it can bind to ssDNA.

Question 10

Outline the process of loading DNA pol III onto DNA.

Answer 10

1. Clamp loader-TauC complex binds to beta-clamp and opens it up
2. Clamp loader-TauC complex places beta-clamp on DNA and closes it
3. Clamp loader-TauC complex is released
4. DNA polymerase III binds to beta-clamp/DNA

Question 11

Outline the steps of replisome assembly.

Answer 11

1. DnaB and DnaG are moving 5’ to 3’ on lagging strands while DnaG adds primers.
2. Clamp loader attaches to DnaB using linker-Tau-C
3. Clamp loader attaches first beta-clamps to lagging strands
4. First beta-clamps start moving 3’ to 5’
5. Clamp loader add second beta-clamp to lagging strand (DNA pol III on stand-by)
6. Clamp loader loops DNA around to load DNA pol II onto beta-clamp
7. Second beta-clamp and DNA pol III begin synthesis of lagging strand and first beta-clamp binds to DNA pol III
8. second beta-clamp and DNA pol III start synthesis of leading strands.

Question 12

Which direction does the replisome move on the leading strand template? Lagging strand?

Answer 12

Leading: 3’ to 5’
Lagging: 5’ to 3’

Question 13

How is it possible for newly synthesized strand of lagging template to be synthesized in 5’ to 3’ direction if the replisome is moving across the lagging strand in the same direction?

Answer 13

Replisome loops the lagging strand, locally flipping the polarity.

Question 14

What happens once the beta-clamp and DNA pol III on lagging strand finish an Okazaki fragment?

Answer 14

They dissociate and rapidly reassemble (with help from clamp loader) upstream of lagging strand to start synthesis of new Okazaki fragment