I . DNA & RNA | 5. Principles of the semiconservative DNA replication; replication fork, leading and lagging strand

Question 1

Characteristics of DNA

Answer 1

1. DNA is a molecule made up of 2 strands, twisted around each other in a double helix shape
2. Each strand is made up of a sequence of bases (A-T, C-G)
3. The 2 strands are complementary: wherever there is a T in one strand, there will be
   an A in the opposite strand
4. Each strand has a 5 prime (‘) end and a 3’ end. The two strands run in opposite
   directions -> this determines how each strand of DNA is replicated

Question 2

1. semiconservative duplication
   a/ What is semiconservative duplication?

Answer 2

It is the mechanism by which DNA is replicated
-> it produces two copies, that each contain one of the original strands (parent) and one new strand (daughter).

Question 3

1. semiconservative duplication
   b/ What are the 3 main activities in Replication in prokaryotes?

Answer 3

synthesis: major activity
builds up the new strand Reading: 3’->5’, Writing: 5’->3’

3’–5’ exonuclease activity: proofreading function:
removes any false nucleotide (no H-bonds)

5’–3’ exonuclease activity: degradation of a strand, that forms a double helix in front of the synthesis

Question 4

1. semiconservative duplication
   c/ What are the elements involving in DNA-synthesis

Answer 4

1. DNA-template
2. Primer: free 3’-OH-group (DNA or RNA)
3. dATP, dCTP, dGTP, dTTP, Mg2+

Question 5

2/ System of DNA polymerase
a/ The role of DNA-Polymerases type I in Prokaryotes

Answer 5

1. consists of a single polypeptide chain (cleavage of one peptide bond -> Klenow-Fragment)
2. synthesize short stretches of DNA during excision repair
3. Remove RNA primers
4. Connecting Okazaki fragments in lagging strand replication.

Question 6

2/ System of DNA polymerase
b/ The role of DNA-Polymerases type III in Prokaryotes

Answer 6

1/ Participate in the synthesis
2/ Have 3’ to 5’ exonuclease activity

Question 7

2/ System of DNA polymerase
c/ The role of DNA-Polymerases type II in Prokaryotes

Answer 7

1/ Repair - has 3’-5’ exonuclease activity
2/ Synthesis- very slow synthesis of DNA and only participates in DNA repair

Question 8

What is the mechanism of replication IN PROKARYOTES

Answer 8

1. Separtaion of the 2 DNA-strands: dnaA recognized the start point: oriC
2. dnaB (+ dnaC) molecules: helicase activity  “initiation bubble”

Question 9

3/ Replication fork
b/ What happen in Replication fork?

Answer 9

The 2 DNA-strands must be unwraped
-> Supercoil structure at flanking regions

Question 10

The role of Topoisomerases

Answer 10

change the spatial structure (supercoil Structure) of the DNA

Question 11

What is positive supercoil?

Answer 11

Direction of the supercoil and that of the turns of the DNA-helix is identical (<10 base pairs/ turn)

Question 12

3/ Replication fork
a/ What are the 4 components at Replication fork

Answer 12

• The DNA helicase unwinds a short segment of parental duplex DNA
• A primase initiates synthesis of an RNA molecule (primer) that is essential for priming DNA synthesis
• The DNA polymerase III initiates the daughter-strand synthesis
• SSBs (single-strand DNA-binding
  protein) binds to ssDNA and prevents remature reannealing of ssDNA to dsDNA

Question 13

Classification of topoisomerase? why?

Answer 13

Question 14

3/ Replication fork - Primosome
c1/ The role of HD-proteins

Answer 14

keep the DNA in single- stranded form

Question 15

3/ Replication fork - Primosome
c2/ The role of HD-proteins

Answer 15

keep the DNA in single- stranded form

Question 16

3/ Replication fork - Primosome
c3/ The role of N-proteins

Answer 16

N-Proteins are attached to dnaB, C

Question 17

3/ Replication fork
c/ 3 Components of primosome

Answer 17

1/ HD-Proteins: keep the DNA in single- stranded form
2/ N-Proteins are attached to dnaB, C
3/ Primase: dnaG: synthesizes the RNA-primer

Question 18

3. Replication fork
   d/ 3 Components of Replisome

Answer 18

1/ Primosome
2/ rep-Proteins: helicase activity
3/ DNA-polymerase III

Question 19

4. Leading strand
   a/ What is the leading strand?

Answer 19

It is the strand in which the direction of DNA synthesis (replication) and the direction of the fork is the same.
- Contain 1 repliosome
- Only one single primer is enough.
- DNA polymerase can do continuously do the job with extrimely high processivity (how long can an enzyme work) -> continuous DNA-synthesis

Question 20

5. Lagging strand
   a/ What is the lagging strand?

Answer 20

• The strand in which
  -> synthesis of the new DNA-strand is opposite to the direction of the replication
  -> several replisomes, Okazaki-fragments

Question 21

5. Lagging strand
   b/ How does DNA synthesis work on lagging strand of prokaryotes?

Answer 21

• DNA polymerase 3 will stop at the previos Okasaki fregment because it doesn’t have 5’ exonucleoase activity.
• Primer must be removed:
  +) Ribonucleases H cleave the RNA of RNA/DNA hybrids that form during replication and repair
  +) DNA polymerase 1- cut the last RNA nucleotide which already connected to DNA, and adding DNA nucleotides
• ligase- connect between the replaced nucleotides with the original nucleotides to create a continous strand, using energy as
  NAD+

Question 22

The role of Ligase

Answer 22

connection of the Okazaki-fargments

Question 23

How does DNA-Ligase work? What is the source of energy?

Answer 23

DNA-Strand break: missing phosphoester bond
-> Connection of the Okazaki-fragments, DNA-repair

Energy: eukaryotes: ATP, prokaryotes: NAD+

Question 24

Replication mechanism of prokaryotes

Answer 24

DNA in prokaryotes: circular
-> replication in both directions

identical number of replisomes on both strands

Question 25

Comparision of prokaryotes and eukaryotes in replication

Answer 25

1/ Mechanism of the replication is similar (leading and lagging strands etc.)

2/ DNA in eukaryotes is much longer and linear
-> several replication bubbles

Question 26

5 types of DNA-polymerases in eukaryotes

Answer 26

Question 27

4. Leading strand
   b/ What happen on leading strand of eukaryotes?

Answer 27

• PCNA- consist of 3 subunits and activated by RFC (replication factor C- homologous to Tao in prokaryotes.
• It form a ring around the template strand of the leading strand and the polymerase epsilon is attach to PCNA (high processivity is ensured, with PCNA the epsilom polymerase is attach to strand and cannot go away).

Question 28

5. Lagging strand
   c/ What happen on lagging strand of eukaryotes?

Answer 28

• Original histones -> to leading strand; new histones -> to lagging strand
• Ligase: energy: ATP

1/ The other strand, the lagging strand, cannot be made in this continuous way since the synthesis occurs in the opposite direction - a 3’ to 5’ direction. The DNA polymerase can therefore only make this strand in a series of small chunks, called Okazaki fragments:
- Each fragment is started with an RNA primer
- DNA polymerase then adds a short row of DNA bases in the 5’ to 3’ direction.
- The next primer is added further down the lagging strand
– Another Okazaki fragment is made, and the process is repeated again
- Once the new DNA has been made, the enzyme exonuclease, removes all the RNA primers from both strands of DNA.
- Another DNA polymerase (delta) then fills in the gaps that are left behind with DNA
- Finally, an enzyme called DNA ligase joins the adjacent fragments of DNA to Form a continuous double strand