I. DNA & RNA | 6. Comparison of DNA replication in pro- and eukaryotic cells: principles, enzymes, proteins

Question 1

Location of replication in prokaryotes and eukaryotes?

Answer 1

Pro - inside the cytoplasm
Eu - Inside the nucleus

Question 2

Similarity in replication mechanism between Prokaryotes and Eukaryotes

Answer 2

Semiconservative Mechanism

Leading Strand (5’-> 3’)
Lagging Strand (3’-> 5’)

Question 3

Difference in replication mechanism between prokaryotes and eukaryotes

Answer 3

Prokaryotes
* Circular DNA
* there’s only one origin of replication, formed of
about 100-200 or more nucleotides
* Replication in both directions
* Okazaki fragments are long (1000-2000 nucleotides long)

Eukaryotes
* Linear and much longer
* Several replication bubbles (over 1000)
each is formed of about 150 nucleotides.
* The replication starts simultaneously at each of these origins
* Okazaki fragments are short in eukaryotes (100-200 nucleotides long)

Question 4

The role of Endonuclease

Answer 4

Cleaves phosphodiester bond within polynucleotide in the middle

Question 5

The role of Exonuclease

Answer 5

Cleaves phosphodiester bond within polynucleotide in the end

Question 6

List the DNA polymerases in prokaryotes

Answer 6

I. Repair- involved in excision repair with both 3’-5’ and 5’-3’ exonuclease activity. So, it can remove the coming primers in Okazaki fragments alone.
Synthesis- Adding nucleotides at the RNA primer. When Pol III holoenzyme is assembled it takes over replication at a highly processive speed and nature. Therefore, Pol I is especially employed at the lagging strand replace the primers sequences to DNA.

II. Repair - has 3’-5’ exonuclease activity; synthesis- very slow synthesis of DNA and only participates in DNA repair

III. Repair- Has 3’-5’ exonuclease activity, synthesis- Replication (MAIN!)

Question 7

List DNA polymerases of eukaryotes

Answer 7

1. alpha: synthesis of initial segment after
   the primase (15–30 nucleotides)
2. beta: DNA-repair (main repair enzyme)
3. gamma: DNA-synthesis in mitochondria
4. delta: synthesis of leading and lagging strands (main synthesis)
5. epsilon: DNA-repair and synthesis too (leading strand)

( Several polymerases do not have exonuclease activity → they form complexes with other exonucleases (3’–5’ proofreading; degradation of RNA-primer). ε δ DNA Polymerases have exonuclease activity)

Question 8

Other proteins and enzymes that are similar in replication in both prokaryotes and eukaryotes

Answer 8

• Ligase
• Topoisomerases (I and II)
  +) Type 1 (topoisomerase 1 and 3): cuts 1 strand, no E source required.
  +) Type 2 (topoisomerase 2 and 4): cuts 2 strands, ATP required.
• Primase

Question 9

Replication in eukaryotes
-> The role of PCNA (proliferating cell nuclear antigen monomer)

Answer 9

forms complex ring with DNA polymerase delta and with epsilon, preventing the polymerases from dissociating from the template

Question 10

Replication in eukaryotes
-> The role of RFC (replication factor C)

Answer 10

function to open the PCNA (proliferating cell nuclear antigen monomer) ring.
-> Catalyzing the leading of PCNA on the DNA 3’ end
- ATP dependent

Question 11

Replication in eukaryotes
-> The role of FEN1

Answer 11

removes the last RNA nucleotides of primer, can remove DNA nucleotides too if H-bond is nor correct

Question 12

Replication in prokaryotes
-> The role of DnaA

Answer 12

a protein that binds to a region of the origin known as the DnaA box.
-> Leading to opening of 13 bp that causes DNA to become negatively supercoiled.
-> This opening of DNA allows protein complexes to enter the replication bubble and bind to the ssDNA. Each complex consist Dnab (helicase) and Dnac (helicase loader).

Question 13

Replication in prokaryotes
-> The role of DnaB (helicase)

Answer 13

DnaB boxes (a region upstream to the DnaA boxes) become unwound. These regions are A-T- rich which facilitates melting that requires ATP
- Helicases are brought in by helicase loading proteins, which inhibit the helicases until they are properly loaded.

(The dnaC protein of Escherichia coli, by forming a complex with the dnaB protein, facilitates the interactions with single-stranded DNA that enable dnaB to perform its ATPase, helicase, and priming functions)

Question 14

Replication in prokaryotes
-> The role of DnaC (loaded helicase)

Answer 14

The loaded helicases open the helicase rings and place in around the ssDNA, enabling primases to synthesize initial primers.

The loaders are then released. -> Two replication forks are assembled and move off in opposite directions.

(The dnaC protein of Escherichia coli, by forming a complex with the dnaB protein, facilitates the interactions with single-stranded DNA that enable dnaB to perform its ATPase, helicase, and priming functions)

Question 15

Replication in prokaryotes
-> The role of SSB
(single strand binding protein)

Answer 15

In order for DNA replication to continue, SSB
(single strand binding protein) is needed to prevent the single strands of DNA from forming any secondary structures and to prevent them from reannealing.

Question 16

Compare the rate of replication between prokaryotes and eukaryotes

Answer 16

Pro - Fast (500 bases/ sec)
Eu - Slow (50 bases/ sec)

Question 17

Compare the time of replication between prokaryotes and eukaryotes

Answer 17

Pro - Occurs almost continuously
Eu - Only at the S phase of the cell cycle