DNA replication initiation (eukaryotes)

DNA replication initiation (eukaryotes)

Origin of replication

  • DNA replication is initiated from specific sequences called origin of replication. Eukaryotic DNA contains many origins of replication. At each origin, a multiprotein origin recognition complex binds to initiate the unwinding of the DNA.
  • In yeast, which is a eukaryote, special sequences known as Autonomously Replicating Sequences (ARS) are found on the chromosomes. These are equivalent to the origin of replication in E.coli.
  • In other eukaryotes, like humans, there does not appear to be a consensus sequence for their origins of replication. Instead, the replication initiation proteins might identify and bind to specific modifications to the nucleosomes in the origin region.

 S.cerevisiae origin of replication (ARS)

  • Yeast ARSs typically consist of 100 to 120 bp of DNA.
  • Yeast ARS contains four regions (A, B1, B2, and B3), named in order of their effect on plasmid stability. When these regions are mutated, replication does not initiate.
  • A region contain a highly conserved 11-bp AT-rich sequence
  • The “A” region is absolutely necessary, while B1, B2, and B3 can increase the replication efficiency. (Mutational analysis for the yeast ARS elements have shown that any mutation in the B1, B2 and B3 regions result in a reduction of function of the ARS element. Mutation in the A region results in a complete loss of function).
Figure 1: Sequence of S. cerevisiae origin of replication (ARS)
  • ORC protein complex (Origin Recognition Complex) is bound at the ARS throughout the cell cycle, allowing replicative proteins access to the ARS.
  • Binding of ORC to ARS probably unwinds the DNA in this region. Interestingly, ORCs also function in regulating transcription.
  • Note that “A” and “T” dominate the replication origin. This is because the A-T pair is linked by two hydrogen bonds while C-T pair by three hydrogen bonds. Therefore, the region dominated by “A” and “T” should be easier to unwind.
  • Melting of DNA occurs within domain B2, induced by attachment of ARS Binding factor 1 (ABF 1) to B3. A1 and B1 domain binds with Origin Recognition Complex.

Eukaryotic cells utilize thousands of origins for the entire genome replication. The use of multiple origins creates a special problem in the timing of replication. The entire genome must be precisely replicated once and only once in each cell cycle so that no genes are left unreplicated and no genes are replicated more than once. How does a cell ensure that replication is initiated at thousands of origins only once per cell cycle?

Answer:

The precise replication of DNA is accomplished by the separation of the initiation of replication into two distinct steps.

Step 1: Licensing of DNA replication

The origins are licensed, meaning that they are approved for replication.

This step is early in the cell cycle when a replication licensing factor attaches to an origin.

Step 2: Initiator proteins cause the separation of DNA strands and the initiation of replication at each licensed origin

The key is that initiator proteins function only at licensed origins. As the replication forks move away from the origin, the licensing factor is removed, leaving the origin in an unlicensed state, where replication cannot be initiated again until the license is renewed.

To ensure that replication takes place only once each cell cycle, the licensing factor is active only after the cell has completed mitosis and before the initiator proteins become active.

All origins are not fired at the same time (See figure 2)

  • Replication does not start at all the different origins at once. Rather, there is a defined temporal order in which these origins fire.
  • Frequently a few adjacent origins open up to duplicate a segment of a chromosome, followed some time later by another group of origins opening up in an adjacent segment.
  • Replication does not necessarily start at exactly the same origin sites every time, but the segments appear to replicate in the same temporal sequence regardless of exactly where the segments are.
Figure 2

The initiation stage of eukaryotic DNA replication includes 3 major events (Figure 5)

  1. Assembly of Pre – RC at origin (G1 phase)
  2. Formation of pre initiation complex (G1 – S phase transition)
  3. Origin firing (S phase)

Event 1: Assembly of pre replicative complex (Pre – RC) – Origin licensing

  • A prereplicative complex (pre-RC) is assembled at each replication origin during the G1 phase of the cell cycle.
  • This is the only period of the cell cycle during which the pre-RC can form and hence this process is known as licensing.
  • However, a licensed pre-RC cannot initiate DNA replication. Rather, it must be activated to do so. But the activation process occurs only during S phase.
  • This temporal separation of pre-RC assembly and origin activation ensures that a new pre-RC cannot assemble on an origin that has already “fired” (commenced replication) so that an origin can only fire once per cell cycle.
Figure 3: Schematic diagram for the assembly of Eukaryotic pre – replication complex

Steps involved in the assembly of eukaryotic pre – replication complex (Pre – RC)

DNA replication begins with the assembly of pre-replication complexes (pre-RCs) at thousands of DNA replication origins during the late M phase or G1 phase of the cell cycle.

Step 1: Binding of ORC to the Origin of replication

  • The ORC is a hexamer of related proteins complex (Orc1 through Orc6) that selects the replicative origin sites on DNA for initiation of replication.
  • ORC binding to chromatin is regulated through the cell cycle.
  • ORC is the functional analog of DnaA protein in E.coli replication initiation.

Step 2: Binding of Cdc6 and Cdt1protein to ORC

  • In late mitosis, Cdc6 protein joins the bound ORC followed by the binding of the Cdt1 protein.
  • Cdc6/Cdc18 together with Cdt1 appears to be an analog of E.coli DnaC (which facilitates DnaB loading).
  • Cdc6 binds to the ORC on DNA in an ATP-dependent manner.
  • Cdt1 is required for the licensing of chromatin for DNA replication.
  • Cdt1 activity during the cell cycle is tightly regulated by its association with the protein geminin, which both inhibits Cdt1 activity during S phase in order to prevent re-replication of DNA and prevents it from ubiquitination and subsequent proteolysis.
  • The combined activities of Cdc6 and Cdt1 bring MCM complexes to replication origins.

 Step 3: Loading of MCM 2 – 7 complex onto the DNA (origin licensing)

  • Mcm protein is the analog of E.coli DnaB helicase (ATP driven helicase).
  • Coordinated action of ORC, Cdc6, and Cdt1 are required to load the six protein minichromosome maintenance (Mcm 2-7) complex onto the DNA.

(With the exception of Cdt1, all of these proteins, Orc1 through Orc6, Cdc6/Cdc18, Mcm2 through Mcm7, as well as E. coli DnaA, DnaB, and DnaC, are AAA+ ATPases).

  • Once the Mcm proteins have been loaded onto the chromatin, ORC and Cdc6 can be removed from the chromatin without preventing subsequent DNA replication.
  • This suggests that the primary role of the pre-replication complex is to correctly load the Mcm proteins

 Event 2: Formation of pre initiation complex

At the G1–S-phase transition, pre-RCs are converted into pre-initiation complexes, in which the replicative helicase is activated, leading to DNA unwinding and initiation of DNA synthesis.

  • This process begins with addition of Mcm10 to the pre-RC

Cdk: Cyclin dependent kinase 2

Ddk: Db4 dependent kinase (heterodimer of the protein kinase Cdc7 with its activating subunit Dbf4)

  • Ddk acts to phosphorylate five of the six MCM subunits (all but Mcm2) so as to activate the MCM complex as a helicase.
  • Ddk together with a Cdk also recruits Cdc45 to the growing initiation complex.

 Cdc45:

  • Required for the formation of an active initiation complex by recruiting key components of the replisome at the replication fork. (Including polymerase α/primase, polymerase ε, PCNA, and replication protein A). DNA replication can then begin.

CDK/DDK-dependent phosphorylation of pre-RC components leads to replisome assembly and origin firing. Cdc6 and Cdt1 are no longer required and are removed from the nucleus or degraded.

GINS: Protein complex essential to the DNA replication process in the cells of eukaryotes. The complex participates in the initiation and elongation stages of replication.

Event 3: Origin firing

Only a subset of origins are activated during any S phase

  • RPA (replication protein A) is a eukaryotic single stranded DNA binding protein (counterpart of E.coli SSB protein).

Long rows of RPA protein will form on a DNA single strand, because each protein molecule prefers to bind next to a previously bound molecule (cooperative binding). This cooperative binding straightens out the DNA template and facilitates the DNA polymerization process.

  • RFC (replication factor C) is a clamp loader required for loading PCNA onto DNA. RFC induces a change in the conformation of PCNA that allows it to encircle DNA
  • The subunits of the RFC complex have significant sequence similarity to the subunits of the bacterial clamp loading ) complex.
  • PCNA is equivalent to the β subunit of DNA polymerase III in E.coli. PCNA is a trimeric protein that forms a closed ring.
Figure 4: Loading of PCNA on to DNA with the help of clamp loader RFC

The full CMG complex is required for DNA unwinding, and the complex of CDC45-Mcm-GINS is the functional DNA helicase in eukaryotic cells.

Cdc45–Mcm–GINS helicase complex

  • Helicases in eukaryotic cells are remarkably complex.
  • Uses energy from ATP hydrolysis to opens up the DNA helix.
  • The catalytic core of the helicase is composed of six minichromosome maintenance (Mcm2-7) proteins, forming a hexameric ring.
  • Loading of Mcm proteins can only occur during the G1 of the cell cycle, and the loaded complex is then activated during S phase by recruitment of the Cdc45 protein and the GINS complex to form the active Cdc45–Mcm–GINS (CMG) helicase at DNA replication forks.
  • Mcm activity is required throughout the S phase for DNA replication.
  • A variety of regulatory factors assemble around the CMG helicase to produce the ‘Replisome Progression Complex’ which associates with DNA polymerases to form the eukaryotic replisome.

The double-stranded DNA is unwound by DNA helicases ahead of polymerases, forming a replication fork containing two single-stranded templates.

The opening of the double helix causes over-winding, or supercoiling, in the DNA ahead of the replication fork. These are resolved with the action of topoisomerases.

Figure 5: Major steps involved in the initiation stage of eukaryotic DNA replication

2 or more polymerases are present at each replication fork

  • DNA polymerase α which contains primase activity, initiates nuclear DNA synthesis by synthesizing an RNA primer, followed by a short string of DNA nucleotides.
  • After DNA polymerase α has laid down from 30 to 40 nucleotides, DNA polymerase δ completes replication of lagging strand and DNA polymerase ε completes leading strand synthesis. (polymerase switching happens once priming of DNA replication occurred)
  • DNA Polymerase δ must interact with proteins PCNA (proliferating cell nuclear antigen) and replication factor C (RFC) to be active. PCNA is the sliding clamp that tethers Pol δ to the DNA to allow processive replication (to prevent the polymerase from falling off the template).
  • DNA replication then proceeds bidirectionally until each replication fork has collided with an oppositely traveling replication fork, thereby completing the replication of the replicon (Elongation and Termination).

Prevention of pre-replication complex re-assembly

  • During each cell cycle, it is important that the genome be completely replicated once and only once. Formation of the pre-replication complex during late M and early G1 phase is required for genome replication, but after the genome has been replicated the pre-RC must not form again until the next cell cycle.
  • In S.cerevisiae, geminin and CDKs (cyclin dependent kinases) prevent formation of the replication complex during late G1, S, and G2 phases by:
  1. Excluding MCM2-7 and Cdt1 from the nucleus
  2. Targeting Cdc6 for degradation by the proteasome
  3. Dissociating ORC1-6 from chromatin via phosphorylation

(An active replication fork will destroy any licensed pre-RCs and unfired initiation complexes in its path, thereby preventing the DNA at such sites from being replicated twice).

Figure 6: Inhibition of licensing system during S, G1, M and G2 phase of cell cycle in eukaryotes

 

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