Replication Initiation

Question 1

When does replication end in bacteria?

Answer 1

When two forks meet at the other side of the circular chromosome.

Question 2

How many ORIs do bacteria have in their chromosome?

Answer 2

1

Question 3

What is oriC?

Answer 3

ORI of E. coli

Question 4

How long is the oriC?

Answer 4

~245 bps long

Question 5

What are the two regions of the oriC?

Answer 5

4 repeats of TTATCCACA
AT-rich region beside it

Question 6

Why is the AT-rich region beneficial in DNA replication?

Answer 6

Only double bonds opposed to the triple bonds in GC. Thus, easier for enzymes to separate dsDNA.

Question 7

What are the four proteins in E.coli required to open the replicaiton bubble?

Answer 7

DnaA (initiator protein), DnaB (DNA helicase), DnaC (helicase loader/inhibitor), SSB protein

Question 8

What is the function of DnaA?

Answer 8

Binds to TTATCCACA sequence and opens up DNA at AT-rich sequence.

Question 9

What is the function of DnaB?

Answer 9

Binds to opened DNA and further unwinds DNA

Question 10

What is the function of DnaC?

Answer 10

Loads DnaB onto opened DNA and releases it at appropriate time.

Question 11

What is the function of SSB proteins?

Answer 11

Bind to and stabilizes ssDNA for other proteins to function properly.

Question 12

Outline the process of opening the replication bubble in E. coli oriC

Answer 12

1. DnaA binds to the TTATCCACA sequence and bends DNA. Stress from bending destabilizes the AT-rich region, opening up part of it.
2. DnaC binds to DnaB and helps it bind to opened DNA
3. DnaC suppresses DnaB function until loading is complete
4. DnaG binds to DnaB
5. DnaC releases DnaB, which moves along the DNA to further open it.
   Whole time, SSB proteins are binding to ssDNA to stabilize it.
6. Dna C carries DnaG while DnaG starts to place primers on ssDNA

Question 13

Why are SSB proteins important?

Answer 13

When single stranded, DNA has a chance to start binding to itself, forming structures that would interfere with synthesis. SSB proteins prevent DNA from forming such structures.

Question 14

Explain the structure of DnaB and how this makes it necessary for DnaC to help it with loading onto DNA.

Answer 14

Donut-shaped made out of multiple subunits. Due to its ring shape, the closed complex is inable to bind to DNA.

Question 15

How many DnaB/DnaC units are loaded onto each strand of ssDNA? Why?

Answer 15

2; each will move in opposite directions to open two replication forks.

Question 16

What is DnaG? What does it do?

Answer 16

DNA primase; adds RNA primers onto ssDNA templates for DNA polymerases.

Question 17

Outline the different interactions the different domains of DnaG have.

Answer 17

One part of protein hypothesized to interact with ssDNA, bringing it to RNA polymerase active site in correct orientation.
C-terminal domain interacts with DnaB

Question 18

Which strand do DnaB/DnaC move along?

Answer 18

Lagging stranf

Question 19

If DnaB/DnaG move 5’ to 3’, does that mean DnaG is synthesizing RNA primers in 3’ to 5’ direction?

Answer 19

No, hypothesized that DnaG has a binding site which bends DNA in the correct orientation for 5’ to 3’ polymerization.