DNA replication in Eukaryotes by VEX

 

DNA replication in eukaryotes

This process occurs in three stages.

  1. Initiation 

  2. Elongation

  3. Termination

 

Initiation

•     DNA replication is initiated from the origins of replication ( Ori ), and eukaryotic nuclear DNA has multiple ori's.

•    Initiation of replication first involves the formation of the pre-replicative complex ( pre-RC ).

•    In yeast ( eukaryotic ) nuclear DNA replication , it starts with the binding of the origin recognition complex ( ORC ) to the ori region which is called autonomously replicating sequence ( ARS ).

• As cells enter the G1 phase of the cell cycle, ATP bound ORC bound to the ARS  recruits ATP-bound Cdc6 & two copies of the Mcm2-7 helicase bound to a second helicase loading protein ( Cdt1 ) to form a protein complex called pre-RC complex.

•  This assembly of proteins triggers ATP hydrolysis by Cdc6, resulting in the loading of the Mcm2-7 helicase & the release of Cdc6 and Cdt1 from the origin.

• Eukaryotic helicase loading does not lead to the immediate unwinding of origin DNA.

•    Helicases that are loaded during G1 phase are only activated to unwind DNA and initiate replication after cells pass from the G1 to the S phase of the cell cycle.

• At the transition of the G1 stage to the S-phase of the cell cycle , two S phase protein kinase , Dbf4/Drf1-dependent kinase ( DDK ) & cycling dependent kinase ( CDK ), phosphorylates to activate Helicase.

• Helicase activation requires the recruitment of helicase-activating proteins ( Cdc45 and GINS ) to helicase , forming Cdc45-MCM-GINS complexes ( CMGs complex ). This transforms the pre-RC into pre-IC

Elongation

• DNA Pol α enzyme first synthesize RNA primers on both the leading and lagging strands. Then adds some deoxyribonucleotides ( initiator DNA ).

•  Once the primer is in place, an event known as polymerase switching occurs, whereby Pol α dissociates from the template and is replaced by Pol δ or ε.

• Pol ε synthesizes DNA on the leading strand, and Pol δ synthesizes the lagging strand.

 •  Completion of lagging strand synthesis requires removal of the RNA primer from each Okazaki fragment. 

In eukaryotes there are 2 models for this function

1.    RNase model

2.      Flap model

RNase model --

• RNase H removes the RNA primer upto the last ribonucleotide.

• FEN 1 ( Flap endonuclease 1 ) removes that last ribonucleotide

• DNA polymerase δ  fills the gap & DNA ligase links the two DNA fragments

Flap model --

• Synthesis of okazaki fragment displaces the RNA primer of the preceding fragment in the form of a flap.

•     The base of flap ( RNA primer ) is cleaved by the enzyme FEN 1.

• DNA ligase links the two DNA fragments.

Replication through Chromatin --

• One of the major differences between bacterial and eukaryotic DNA is that eukaryotic DNA is complexed with DNA-binding proteins, existing in the cell as chromatin

• Before DNA polymerases can begin synthesis, nucleosomes and other DNA-binding proteins must be removed away to allow the passage of replication proteins.

•    In order to re-create nucleosomal chromatin on replicated DNA, the synthesis of new histone proteins is tightly coupled to DNA synthesis during the S phase of the cell cycle.

• The assembly of new nucleosomes is carried out by chromatin assembly factors (CAFs) that move along with the replication fork

Termination

•    Unlike the closed, circular DNA of bacteria and most bacteriophages, eukaryotic chromosomes are linear.

•    The presence of linear DNA ends ( telomeres ) on eukaryotic chromosomes creates a problem called the end replication problem.

End replication problem --

•   As the lagging-strand replication machinery reaches the end of the chromosome, at some point, primase no longer has sufficient space to synthesize a new RNA primer. This results in incomplete replication & a short ssDNA region at the 3’ end of the lagging-strand DNA product.

•  When this DNA product is replicated in the next round, one of the two products will be shortened and will lack the region that was not fully copied in the previous round of replication.

Solution of the End Replication Problem 

•  Telomerase Solves the End Replication Problem by Extending the telomere of the lagging strand template. • The telomerase contains RNA template that binds to the 3' end of the lagging-strand template.  • The telomerase then adds deoxyribonucleotides using the RNA molecule as a template.  • After extension of 3' end of the lagging strand template, DNA pol α ( primase ) catalyzes synthesis of new Okazaki fragments on this extended template strand.