Bacterial replication

Question 1

Which strand is synthesized continuously?

Answer 1

Leading strand

Question 2

Which strand is synthesized discontinuously and forms Okazaki fragments?

Answer 2

Lagging strand

Question 3

What is the sequence of the origin of replication (ori)?

Answer 3

sequence with high A:T content where proteins will bind, separate the double stranded DNA, and produce 2 replication forks

Question 4

How many ori per chromosome?

Answer 4

In prokaryotic: 1
In eukaryotic: hundreds/thousands
Eukaryotic requires synchronization where prokaryotic does not

Question 5

What 4 things are required to prepare the ori for replication in E. coli?

Answer 5

3 core proteins: DnaA, DnaB (Helicase), DnaC, and 1 accessory protein SSB (Single stranded DNA binding protein)

Question 6

Function of DnaA

Answer 6

DNA binding domain specific to ori
Multimerization with DNA wrapping relaxes DNA at A:T region

Question 7

Function of DnaB

Answer 7

Hexameric helicase that unwinds dsDNA into ssDNA

Question 8

Function of DnaC

Answer 8

assists with loading helicase

Question 9

Function of SSB

Answer 9

keeps the two ssDNA strands apart and prevents reannealing

Question 10

How does DnaA protein recognize origin of replication?

Answer 10

• One helix in HTH fits nicely in the major groove
• has some proteins that interact with minor groove to add stability
• part of HTH interacts with phosphate backbone (not sequence specific)

Question 11

Describe base pair interactions at the consensus sequence (ori)

Answer 11

not many mutations, and is less mutable because function is essential to survival (unless co-evolution between consensus sequence and protein)

Question 12

describe initiation at the Ori

Answer 12

-specific binding at DnaA sites leads to cooperative coating of approx 20 DnaA proteins to the right half of the origin around DnaA (requires ATP)
- DnaA multimerizes and causes DNA to wrap around it and bind more DNA, while removing twists from DNA and allowing it to be opened by spontaneously dentaturing

Question 13

How does helicase assemble

Answer 13

• 6 DnaC bound to 6 DnaB allow assembly of single DnaB hexameric ring around each strand
• DnaC is released and helicase will use ATP to move in a 5’ to 3’ direction
• because the DNA is encircled, the enzyme is highly processive meaning it won’t fall off

Question 14

how does helicase work?

Answer 14

• hexamer works as 3 pairs with each subunit on opposite sides
• each subunit extends a peptide loop which can contact phosphate backbone of coiled ssDNA
• ATP binding and hydrolysis drive each protein through 3 shape conformations (ATP-bound : extended, ADP-bound: middle, Empty: low
• each opposing pair generates some force so when they work in sequence there is continuous progression that pulls DNA apart

Question 15

What is primase?

Answer 15

5’ to 3’ DNA dependent RNA polymerase that adds fragments on the lagging strand

Question 16

How does primase work?

Answer 16

• each hexameric ring binds 2/3 molecules of primase
• as helicase travels, primase produces an RNA primer (10-14nt long) every 1500-2000 nt

Question 17

what is found ahead of bacterial replication fork?

Answer 17

-helicase unwinding causes positive supercoiling ahead of replication fork
-topoisomerase II and gyrase convert positive supercoil into negative supercoil by forming double-strand break and removing two twists
- double strand break occurs 450 000 times in E. coli genome (4.5Mb) because one turn = 10 bp

Question 18

Pol III classifications

Answer 18

core enzyme: minimum subunits needed to synthesize DNA
holoenzyme: core plus extras

Question 19

What 3 things make up the Pol III core enzyme structure?

Answer 19

heterotrimer contains subunits
alpha: polymerase with 5’ to 3’ activity
epsilon: proofreader with 3’ to 5’ exonuclease activity
theta: stabilizes epsilon and increases exo rate

Question 20

what makes up the pol III holoenzyme?

Answer 20

• Pol III core (x2-3: one for each strand + one for Okazaki fragments)
• sliding clamp
• clamp loader

Question 21

Function of pol III core in holoenzyme

Answer 21

extends the DNA copy from the RNA primer

Question 22

Structure and function of sliding clamp

Answer 22

dimer that acts as a mobile tether and prevents pol III from releasing target DNA therefore increasing processivity

Question 23

Structure and function of clamp loader

Answer 23

ATPase that repositions the sliding clamp when initiating a new Okazaki fragment and synchronizes leading and lagging strands

Question 24

Pol III processivity

Answer 24

-slow synthesis with only 10-15 nucleotides before separation from DNA
- addition of sliding clamp increases is to about 500 000 for the leading strand
- about 500-1000 nucleotides added per second

Question 25

pol III error rate

Answer 25

• approx. 1 in 10 000 nucleotides will be incroporated incorrectly
• of those, only 1 in 1000 will not get removed by 3’ to 5’ exonuclease
• combined error rate is 1 in 10^7
• about 30/100 correct nucleotides are also removed by 3’ to 5’ exonuclease (false pos.)

Question 26

rough math on E. coli

Answer 26

• E. coli genome is 4.5 Mb
• single origin gives bidirectional replication fork = 2.25 Mb of synthesis in each direction
  -pol III goes 500-1000 nt/sec
• replication takes at least 2,250,000/1000 seconds
• 2250 seconds = 37.5 minutes but we know E. coli divides in 20 minutes

Question 27

How does E. coli replicate it’s genome fast enough for cell division?

Answer 27

“born pregnant” with overlapping cell cycles and multifork replication
- up to 12 replication forks in one cell under optimal conditions
- replication initiates 3 times before one is finished
- allows E. coli to replicate in 20 minutes

Question 28

does the leading strand require a primer?

Answer 28

yes, it comes from the first primer synthesized when replication begins

Question 29

What happens to the primer on the lagging strand?

Answer 29

Pol III does not remove or displace the RNA primer on the lagging strand
- RNAse H or DNA pol I can remove the primer

Question 30

What is RNAse H?

Answer 30

can cleave bond between adjacent ribonucleotides in RNA:DNA hybrids only

Question 31

What is Pol I?

Answer 31

first polymerase discovered and described in 1956, was noted that there was too much in a cell (hundreds of copies) and it worked too slowly (10nt/sec) to be the primary polymerase

Question 32

What type of activity does pol I have?

Answer 32

5’ to 3’ pol
5’ to 3’ exo
3’ to 5’ exo (error check)

Question 33

Pol I function in the lab

Answer 33

used for lagging strand cleanup by nick translation
- can be used in the lab to synthesize labeled DNA

Question 34

Ligase overall function

Answer 34

• After the primer is removed by RNAse H or Pol I the nicks on the lagging strand must be sealed so that the Okazaki fragments are attached to eachother continuously
• Ligase mediates the reaction using NAD+ or ATP to add AMP to the 3’ end and then swab that bond with the 5’ end phosphate to release AMP
• if the 5’ phosphate has been removed there is no ligation - so dephosphorylated DNA cannot be sealed on its own

Question 35

what is the solution to seal dephosphorylated DNA

Answer 35

if the 5’ nucleotide is removed, the next nucleotide has a phosphate. DNA pol I can move it one base forward and then it can be sealed by ligase