DNA Replication in Eukaryotes (initiation-elongation-termination)

Molecular Biology notes on DNA Replication in Eukaryotes (initiation-elongation-termination) DNA polymerase Mechanism of DNA replication and eukaryotic Protein

1. ABG -402 INTRODUCTORY MOLECULAR GENETICS

2. GROUP 2 M BAQIR LAIBA ARIF KANWAL AKBAR ESHA IQBAL NAVEED NAZAR Submitted to : Mam Warda Sarwar

3. DNA Replication in Eukaryotes

4. Replication in E. coli begins at only one site along the single, circular chromosome. Eukaryotes have thousand times bigger DNA yet their polymerases incorporate nucleotides into DNA at much slower rates. Eukaryotic cells replicate their genome in small portions, termed replicons. Each replicon has its own origin from which replication forks proceed outward in both directions. In a human cell, replication begins at about 10,000 to 100,000 different replication origins. Approximately 10 to 15 percent of replicons are actively engaged in replication at any given time during the S phase of the cell cycle Replication in Eukaryotic Cell

5. DNA polymerase Five “classic” DNA polymerase designated as DNA polymerase α, β, γ, δ, and ε . Polymerase γ replicates mitochondrial DNA Polymerase β functions in DNA repair DNA polymerase α and δ are involved in replication (nuclear) DNA polymerase α is typically a multi-subunit enzyme One subunit has a primase activity The largest subunit contains the polymerization activity

6. DNA unwinding and DNA synthesis are assembled in multistep process. There are three major steps that are involved in DNA replication . ➤ Initiation ➤ Elongation ➤ Termination Mechanism of DNA Replication

7. All replication systems require helicases, single‐stranded DNA‐binding proteins, topoisomerases, primase, DNA polymerase, sliding clamp and clamp loader, and DNA ligase Eukaryotic proteins

8. Initiation Initiation of DNA replication is the first stage of DNA synthesis and it starts from specific sequences called origin of replication and eukaryotic cells have multiple origin of replication . Pre-replication Complex Pre-replication complex forms at origin of replication during G1 phase . In most eukaryotes it is composed of ORC Cdc6,Cdt1 and MCM . ORC stands for origin replication complex .It recognizes and binds with origin of replication. All the other proteins Cdc6,Cdt1 and MCM bind with ORC and this whole complex is known as pre- initiation complex. First of all Cdc6 known as cell division cycle 6 binds with ORC.After that Cdt1 which is known as chromatin licensing DNA replication factor 1 helps minichromosome maintenance protein complex to bind with ORC. Initiation Complex The main purpose of Cdc6 and Cdt1 is to load MCM on DNA. After the loading of MCM these proteins are phosphorylated for the activation of initiation complex. This complex is formed during the transition of G1 phase to S phase. Cyclic dependent kinase CDK and Dbf4 protein kinase phosphorylate Cdc6 and Cdt1 and make this complex active for the unwinding of DNA.

9. Activator Complex After the activation of initiation complex two more complex factors Cdc45 and GIN activates the MCM helicase activity. Therefore this complex is known as activator complex which is formed during the S phase of cell cycle. It unwinds the DNA and replication fork is formed.

10. Elongation Replication Fork in humans, synthesis of new DNA occurs at the rate of 300 nucleotides per minute . Once initiation complex is formed the cell is passed into S Phase and replication fork is formed. This complex then becomes a replisome. The eukaryotic replisome complex is responsible for coordinating of DNA replication. It consists of polymerase enzymes ,clamp loader proteins, Topoisomerase and all other important factors that help in replication of DNA strands. Replication Protein A (RPA) RPA is major protein that binds to single stranded DNA in eukaryotic cells.RPA prevents DNA from winding back on itself or forming secondary structures. It keeps the DNA unwound for polymerase to replicate it.

11. Leading Strand Leading strand is template strand and it is replicated in the same direction as the movement of replication fork. Replication is continuous along the leading strand. It occurs from 5' to 3' direction. Many polymerase enzymes and proteins are involved in the replication of leading strands . Lagging strand DNA replication on the lagging strand is discontinuous and it is away from the replication fork. Although newly formed DNA strand also run in 5' to 3' direction.

12. Termination In eukaryotes DNA polymerase cannot replicate the terminal DNA segment of lagging strand of linear chromosomes. The terminal region of DNA is known as telomere. Telomeres must have unique features and enzymes that facilitates replication .

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