Lecture 2- duplication

Question 1

Origin of replication in E.coli

Answer 1

oriC

This is 245 base pairs, 5 repeats of a 9bp sequence that is a binding site for the initiator protein DnaA

Question 2

Name 2 DNA bending proteins which separate the strands

Answer 2

IHF and Fis

Question 3

DNA unwinding element (DUE)

Answer 3

AT rich sequence adjacent to where DnaA assembles. This is where DNA is pulled apart for replication.
When DUE melts (base pairs pulled apart) DnaA becomes compact to force the strands apart

DUE is where the strand opens to form the initial bubble

Question 4

DnaB - function and problem

Answer 4

Opens the replication form

Ring shaped protein - difficult to load onto a long molecule of DNA that is also circualr

Question 5

How is DnaB loaded onto DNA?

Answer 5

DnaC-ATP ring binds the DnaB ring and opens it for loading
DnaC-ATP will direct DnaB to the origin opened by DnaA
Binding of the DnaBC complex to DnaA promotes assembly of two DnaB rings, one for each replication fork
ATP hydrolysis releases DnaC leaving DnaB bound to the DNA

Question 6

SSB

Answer 6

Single stranded DNA binding protein

Accessory pigment

Question 7

Function of SSB

Answer 7

Stops unwound parent strands from re-annealing
Prevents intramolecular strand annealing- separated single strand that may have complementary sections may anneal to form a hair pin shape, this results in deletions
Protects DNA strands from degradation or damage
Acts as an assembly point for multiple proteins involved in DNA replication
Can block access of proteins involved in DNA replication

Question 8

DNA gyrase - topoisomerase II

Answer 8

As DnaB unwinds parental strands ahead of the replication fork the unwound parental DNA can coil up (supercoiling) and result in a clock to fork progression

DNA gyrase alleviates supercoiling by cutting the two strands of DNA and passing a strand through the break before resealing the cut strands.

Question 9

What do all DNA polymerases have in common?

Answer 9

A proof reading ability so they can check errors when nucleotides are inserted.

Question 10

Fidelity

Answer 10

The faithful copying of DNA and how accurate it is

Question 11

DNA polymerase II and its proofreading subunit

Answer 11

DNA polymerase II has an epsilon proofreading subunit with exonuclease activity.
Without this an incorrect nucleotide would be inserted every one in 7 million base pairs.

Question 12

Function of DNA polymerase I

Answer 12

Removes RNA primer and replaces with DNA

single subunit and has 5’ to 3’ exonuclease activity

Question 13

Function of DNA polymerase II

Answer 13

Fills in gaps following repair of DNA damage

Question 14

DNA polymerase III

Answer 14

Chromosome replication

Initiates DNA synthesis by extending an RNA primer

Question 15

The core polymerase has… (3 points)

DNA polymerase III

Answer 15

A separate epsilon subunit that specifies the 3’ - 5’ exonuclease (for proof reading)
two alpha subunits
Two tau subunits that attach the alpha polymerase subunits and hold them together

Question 16

DNA polymerase III

Gamma complex

Answer 16

Gamma complex/clamp loader 
Binds tau (T) and loads the beta sliding clamp onto the parental strands

Question 17

DNA polymerase III

Beta clamp

Answer 17

Ring (homodimer) that encircles the parental strand on each daughter template to ensure the polymerase does not fall off during DNA synthesis (enhances processivity)

Question 18

What allows opening of the beta clamp ring?

Answer 18

gamma complex bound to ATP while ATP hydrolysis allows clamp release and ring closure

Question 19

Priming different strands

Answer 19

The leading strand is primed one at the replication origin (twice as there are two bidirectional forks).
On the lagging strand DnaG primase syntheises an RNA primer for every Okazaki fragment as synthesis is directed on a parental loop and synthesised discontinuously.

Question 20

Beta clamps and RNA primers

Answer 20

A new B clamp is laoded on each RNA primer by the clamp loader (gamma complex).
As new okazaki fragments are formed the clamp loader (gamme) opens a new clamp (Beta) and the helicase (DnaB) recruits primase (DnaG) to the replication fork to initiate the next fragment.

Question 21

What happens after the synthesis of the RNA primer?

Answer 21

The clamp loader displaces primase (DnaG) and loads the clamp onto the new primer.

Question 22

How long does it take E.coli to replicate?

Answer 22

DNA polymerase III works at 900 nucleotides per second. It should take 42 minutes for the chromosomal DNA to be replicated but it is less than this as the next round of replication is initiated before the firsst round has been completed

Question 23

Summarise all the key component features of bacterial DNA replication (12)

Answer 23

Single origin of replication 
DnaA opens up DNA
initiation coordinated with cell cycle 
DnaC loads DnaB 
DnaB unwinds DNA at the fork 
SSB keeps unwound strands apart 
DnaG makes RNA primer
gamma complex loads the Beta clamp 
B clamp keeps polymerase on DNA
DNA polymerase is Pol III
RNA primer removed by Pol I 
DNA ligase seals the nick