Beginning Of DNA Replication In E. Coli

Question 0

How to identify host replication origin

Answer 0

1. Digest host DNA and plasmid with restriction enzymes
2. ligase =insert host fragments into plasmid with ampR
3. Transform into E. coli
4. Select for colonies on amp media- the plasmids recombined with host replication origins can be replicated into colonies
5. Surviving plasmids can be isolated from bacteria
6. Sequence DNA to identify host replication origin

Question 1

Replication Origin in E. coli

Answer 1

oriC

Question 2

Y- arc indicates

Answer 2

Replication origin outside digested fragment

Therefore replication fork

Y with 3 equal arm lengths at top of gel (shape least streamlined)

Linear DNA at bottom right of gel = smallest size, linear shapes

Question 3

Bubble arc indicates

Answer 3

Replication origin in middle of fragment (rare)

No Y fork, only bubble or linear

Linear DNA at bottom right (replicated or unreplicated no fork or bubble; linear w small size)

Question 4

Bubble-to- Y arc indictes

Answer 4

Replication origin is within digested fragment, but not in the centre

Arc breaks when bubble reaches end of fragment to produce Y DNA replication fork

Linear DNA at bottom right (replicated or unreplicated no fork or bubble; linear w small size)

Question 5

Repetitive sequences in oriC

Answer 5

13-mer and 9-mer

Question 6

Methylated sites?

Answer 6

GATC (adenine methylated) by Dam methylase

Question 7

oriC- 9-mer

Answer 7

DnaA initiator protein binding sites
Consensus sequence
4: R1 R2 R3 R4
Different directions

Question 8

OriC: 13-mer

Answer 8

3 in oriC
AT rich
Direct repeats (same direction)
Locus of initial DNA strand separation

Question 9

OriC : Sites methylated by Dam methylase?

Answer 9

11 GATC- A methylated

Dam methylase = DNA adenine methyltransferase

Question 10

2D agarose gel electrophoresis analysis of replication origins

Answer 10

1. Digest plasmid into fragments
2. 2D gel electrophoresis size (low voltage) and shape (high voltage)

DNA replication forks travel more slowly through the gel than unreplicated or completely replicated fragments bc less streamlined

6. Southern blotting = DNA in gel transferred to nitrocellulose and probed with radioactive 32P DNA fragment that hybridizes to region if interest
7. Y- arc, bubble-to-Y arc or bubble arc = identify location of replication origin relative to probe/ fragment

Question 11

3 mech of control

Regulation of E. coli replication initiation

Answer 11

1. Methylation state of DNA and binding of SeqA
2. DnaA.ATP levels- hydrolysis can take 30 mins for ADP-> ATP
3. Txn in origin region (RNAP tension on DNA)