Initiation of DNA Replication

The initiation of DNA replication in eukaryotes is a highly regulated and multi-step process that ensures DNA is copied accurately and only once per cell cycle. This process involves the assembly of several protein complexes at specific regions of the DNA called origins of replication. Let's break down the initiation phase in detail:

Steps Involved in the Initiation of DNA Replication in Eukaryotes

1. Origin Recognition and Licensing

• Origin Recognition Complex (ORC): The initiation process begins with the binding of the Origin Recognition Complex (ORC) to the DNA at replication origins. ORC is a multi-subunit protein complex that serves as a platform for the recruitment of additional factors.

• Licensing the Origin: The ORC recruits two key proteins, Cdc6 and Cdt1, which are essential for loading the Mini-Chromosome Maintenance (MCM) complex onto the DNA.

• MCM Complex Loading: The MCM complex (a helicase) is loaded onto the DNA as an inactive double hexamer around the origin during the G1 phase of the cell cycle. This step is crucial for "licensing" the origin, meaning it prepares the DNA for replication but doesn't yet start the replication process.

• At this point, the origin is considered licensed, but replication has not yet begun.

2. Activation of the Pre-Replication Complex

• As the cell enters the S-phase, several kinases become activated, primarily Cyclin-Dependent Kinase (CDK) and Dbf4-Dependent Kinase (DDK).

• CDK and DDK Activation: These kinases phosphorylate components of the pre-replication complex (pre-RC), particularly MCM, Cdc6, and Cdt1, leading to the disassembly of Cdc6 and Cdt1 and the activation of MCM.

• Cdc45 and GINS Complex: The activation of MCM helicase also requires additional proteins, such as Cdc45 and the GINS complex. Together, they form the active helicase complex known as CMG (Cdc45-MCM-GINS).

• The activation of the CMG complex leads to the unwinding of DNA, creating single-stranded DNA (ssDNA) regions.

3. Formation of the Replication Fork

• Single-Stranded DNA Binding Proteins (RPA): Once the DNA is unwound, Replication Protein A (RPA) binds to the exposed single-stranded DNA to prevent it from reannealing or forming secondary structures.

• Primase and DNA Polymerase α: The enzyme DNA polymerase α-primase complex is recruited to the replication fork to synthesize a short RNA-DNA hybrid primer. This primer provides a starting point for DNA synthesis.

• Leading and Lagging Strand Synthesis: After the primer is laid down, DNA polymerase δ and DNA polymerase ε are recruited for the synthesis of the lagging and leading strands, respectively.

ORC (Origin Recognition Complex): Binds to origins and recruits other factors

Cdc6 and Cdt1: Help load the MCM helicase onto DNA

MCM Complex: Acts as the helicase that unwinds DNA

CDK and DDK: Kinases that activate the pre-replication complex

Cdc45 and GINS: Assist MCM in helicase activation, forming the CMG complex

RPA (Replication Protein A): Stabilizes single-stranded DNA

DNA Polymerase α-Primase: Synthesizes RNA-DNA primers

Elongation of DNA Replication

The elongation phase of DNA replication in eukaryotes is the process where the bulk of DNA synthesis occurs, following the initiation phase. During elongation, the replication machinery (the replisome) efficiently synthesizes new DNA strands by adding nucleotides to the growing DNA chain. This phase involves multiple enzymes and proteins to ensure high-speed and high-fidelity DNA replication. Let's break down the elongation process step by step.

Key Steps of DNA Elongation in Eukaryotes

1. Unwinding the DNA

• Helicase Activity (CMG Complex):

  • The CMG complex (formed by Cdc45, MCM helicase, and GINS) continues to unwind the parental DNA double helix at the replication fork.

  • This unwinding generates two single-stranded DNA templates for new strand synthesis.

2. Stabilizing Single-Stranded DNA

• Replication Protein A (RPA):

  • As the DNA unwinds, single-stranded DNA (ssDNA) regions are exposed. These ssDNA regions are prone to forming secondary structures or getting degraded.

  • RPA binds to the ssDNA, stabilizing it and preventing the formation of secondary structures that could impede the replication process.

3. Synthesis of RNA Primers

• DNA Polymerase α-Primase Complex:

  • Primase, a component of the DNA polymerase α-primase complex, synthesizes a short RNA primer (approximately 10 nucleotides).

  • DNA polymerase α then extends this RNA primer with a short stretch of DNA (around 20-30 nucleotides), creating an RNA-DNA hybrid primer.

  • This primer is required for DNA polymerases to begin synthesizing new DNA.

4. Leading Strand Synthesis

• DNA Polymerase ε:

  • On the leading strand, which is synthesized continuously, DNA polymerase ε takes over after the initial primer is laid down by DNA polymerase α.

  • DNA polymerase ε synthesizes the new strand in the 5' to 3' direction, following the replication fork as it opens.

5. Lagging Strand Synthesis

• DNA Polymerase δ:

  • The lagging strand is synthesized in short, discontinuous fragments called Okazaki fragments (about 100-200 nucleotides long in eukaryotes).

  • Each Okazaki fragment begins with a new RNA primer synthesized by the DNA polymerase α-primase complex.

  • DNA polymerase δ then extends these primers to create the Okazaki fragments.

  • As the replication fork progresses, new primers are laid down, and additional Okazaki fragments are synthesized.

6. Sliding Clamp (PCNA) and Clamp Loader (RFC)

• Proliferating Cell Nuclear Antigen (PCNA):

  • To enhance the processivity (the ability of DNA polymerase to synthesize long stretches of DNA without dissociating), PCNA (a sliding clamp) encircles the DNA and tethers DNA polymerases δ and ε to the DNA.

  • Replication Factor C (RFC) acts as a clamp loader, helping to load PCNA onto the DNA.

7. Removal of RNA Primers and Joining of Okazaki Fragments

• RNase H and FEN1:

  • Once the lagging strand Okazaki fragments are synthesized, the RNA primers need to be removed.

  • RNase H and Flap Endonuclease 1 (FEN1) remove the RNA primers.

• DNA Ligase I:

  • DNA ligase I seals the gaps between adjacent Okazaki fragments by forming phosphodiester bonds, thus creating a continuous DNA strand.

Termination of DNA Replication

The termination of DNA replication in eukaryotes is the final stage of the replication process, where the replication machinery stops synthesizing DNA and the newly synthesized DNA strands are separated and processed. Unlike prokaryotes, which have a single circular chromosome with defined termination sites, eukaryotic chromosomes are linear and more complex, making termination a coordinated process involving multiple factors. Here's a detailed explanation of how termination occurs in eukaryotic cells:

Key Steps in Termination of DNA Replication

1. Convergence of Replication Forks

• Eukaryotic chromosomes have multiple origins of replication, which means that replication forks progress bidirectionally from these origins.

• Termination occurs when two replication forks converge from adjacent origins. At this point, the DNA between the forks is fully replicated.

• As the forks converge, the helicases (CMG complex) are inactivated, and the unwinding of DNA stops.

2. Removal of RNA Primers

• On the lagging strand, replication produces short Okazaki fragments, each initiated by an RNA primer.

• The RNA primers are removed by RNase H and Flap Endonuclease 1 (FEN1).

• DNA polymerase δ fills in the gaps with DNA, and DNA ligase I seals the nicks to create a continuous strand.

3. Decatenation of DNA Strands

• After synthesis is complete, the newly replicated DNA molecules may become entangled or catenated (interlinked), especially near the ends of the chromosome.

• Topoisomerase II is an enzyme that helps resolve these tangles by creating temporary double-stranded breaks in the DNA, allowing the strands to pass through each other and then resealing the breaks.

• This process, known as decatenation, ensures that the two daughter DNA molecules are fully separated.

4. Dealing with Telomeres

• Eukaryotic chromosomes have telomeres, which are repetitive DNA sequences at their ends that protect against degradation and prevent chromosome fusion.

• The replication of telomeres presents a unique challenge known as the end-replication problem because the lagging strand cannot be fully replicated to the very end.

• To solve this, an enzyme called telomerase extends the 3' end of the parent strand by adding repetitive nucleotide sequences.

• Telomerase contains an RNA template that it uses to synthesize DNA repeats, thus preventing chromosome shortening with each cell division.

• After telomerase extends the parent strand, DNA polymerase α-primase synthesizes an RNA primer, allowing DNA polymerase to fill in the complementary strand.