11. Principle of replication. Enzymes involved in the replication and their spatial organization. DNA polymerases.

Question 1

Principle of replication

Answer 1

-Replication is the doubling of DNA by matrix synthesis.
base on the complementary recognition of nucleotides.

-Each daughter DNA molecules consist of one old and one new strand, mechanism of replication - semiconservative.

Replication takes place during the S-phase of he cell cycle

Question 2

What is necessary for replication?

Answer 2

-DNA matrix
-4 deoxyribonucleotide triphosphates- ATP, GTP, CTP, TTP.
Family of enzymes:
-Topoisomerase
-Primase(primer)
-Helicases
-DNA polymerase
-DNA ligase
-Telomerase

Question 3

Initiation

Answer 3

DNA single unit replication = Replicon

The initiation of DNA replication within a replicon occurs at fixed point known as the ORIGIN.
-At the Origin, 2 DNA strands must be separated to serve as templates. this happens with help of enzyme HELICASE.

Circular DNAs in prokaryotes have a single origin - the entire DNA Molecule in a replicon.
In contrast, the long linear DNA in eukaryotes chromosomes has multiple origins-multiple replicons.

The unwound sections of parental DNA strands are now available to be templates for the synthesis of new complementary DNA strands.
HOWEVER, the enzyme that synthesizes DNA cannot initiate synthesis of a polynucleotide, it can only add DNA nucleotides to the end of existing chain.

Therefore, initial nucleotide chain is produced, called PRIMER.
Synthesized by enzyme called primase.

Question 4

Elongation

Answer 4

General Mechanism:

Enzyme: DNA polymerase.
Direction: 5’ to 3’.
Nucleotides: Added as triphosphates, linked by phosphodiester bonds.
Complementary Binding: Spontaneous base pairing; hydrogen bonds form.

Leading Strand:
Synthesis: Continuous, follows replication fork.
Primer: Only one needed.

Lagging Strand:
Synthesis: Discontinuous, forming Okazaki fragments.
Fragment Joining: Nuclease removes primers, DNA polymerase fills gaps, DNA ligase joins fragments.
Replication Fork:

Direction: Bidirectional from origin (ORI)

Question 5

Termination

Answer 5

Replication continues until it covers the entire DNA molecule.

From one DNA molecule, two are obtained, each of them having one old and one new strand.

In prokaryotes, the DNA is circular, so there is no problem at the end of the molecule.

Question 6

Enzymes involved in replication and their spatial organization.

Answer 6

-DNA polymerase
-Topoisomerases
-Helicases
-Primase
-DNA ligase
-Telomerase

Helicase:
-Function: Separates parental DNA strands by breaking hydrogen bonds using ATP.

-Structure: 6 subunits, forms a ring around the lagging strand.

-Activity: Unwinds DNA bidirectionally from the origin; needs at least two helicases.

-Attachment: Uses a special protein, released when primase arrives.

Initiation:
-Initiator Protein: Recognizes the origin of replication (e.g., Cdc6 in eukaryotes) and signals other proteins.

Process: Initiator binds to origin, helicase unwinds DNA.

Primase:
-Function: Initiates replication.
-Activity: Synthesizes short RNA primers (about 10 nucleotides) at the 5′ ends of the leading strand and each Okazaki fragment.

Topoisomerase:
Function: Relieves the strain ahead of the replication fork by breaking, swiveling, and rejoining DNA strands.

Question 7

DNA Polymerase

Answer 7

Function: Synthesizes DNA by adding nucleotides to the 3′ end.

Types:

Prokaryotes:
Pol I: DNA repair.
Pol III: Main replication enzyme.

Eukaryotes:
Pol α: Part of primase.
Pol β: DNA repair.
Pol γ: Mitochondrial DNA replication.
Pol δ: Leading strand synthesis.
Pol ε: Lagging strand synthesis.