Process of Replication in Eukaryotes / Biology / notes
- 1. Process of Replication in Eukaryotes Eukaryotic DNA replication is a highly regulated and complex process that ensures the accurate duplication of the entire genome. The replication occurs during the S phase of the cell cycle and involves several key steps: 1. Initiation Origin Recognition: DNA replication begins at specific sequences called origins of replication. Eukaryotic chromosomes contain multiple origins to allow rapid replication of large genomes. Pre-replication Complex Formation (Pre-RC): During the G1 phase, the Origin Recognition Complex (ORC) binds to the origin, recruiting other proteins such as Cdc6, Cdt1, and the Mini-Chromosome Maintenance (MCM) complex (a helicase). This forms the pre- replication complex. Activation of Replication: In the S phase, cyclin-dependent kinases (CDKs) and Dbf4-dependent kinase (DDK) activate the MCM helicase, allowing it to unwind DNA, creating replication forks. This marks the transition from the pre-replicative to the replicative state. 2. Unwinding of DNA Helicase Activity: The MCM helicase unwinds the double-stranded DNA into two single strands, creating a replication fork. Single-Strand Binding Proteins (RPA): Replication Protein A (RPA) binds to the single-stranded DNA to prevent it from reannealing or forming secondary structures. 3. Primer Synthesis Primase: A primase, a component of the DNA polymerase α complex, synthesizes a short RNA primer complementary to the single-stranded DNA. This primer provides a 3’ hydroxyl group for the addition of nucleotides by DNA polymerases. 4. Elongation Leading Strand Synthesis: On the leading strand, DNA polymerase ε synthesizes DNA continuously in the 5’ to 3’ direction, following the replication fork.
- 2. Lagging Strand Synthesis: On the lagging strand, DNA polymerase δ synthesizes DNA in short fragments called Okazaki fragments. Each fragment is initiated by an RNA primer synthesized by DNA polymerase α, and the DNA is synthesized in the 5’ to 3’ direction away from the replication fork. 5. Removal of RNA Primers and Ligation RNase H and FEN1: RNA primers are removed by RNase H and Flap Endonuclease 1 (FEN1). DNA Polymerase δ: After the removal of RNA primers, DNA polymerase δ fills the gaps with DNA nucleotides. DNA Ligase: DNA ligase seals the nicks between the Okazaki fragments, forming a continuous DNA strand. 6. Termination Replication continues until two replication forks meet, or the replication machinery reaches the end of the chromosome (telomeres). Telomere Replication: The enzyme telomerase adds repetitive sequences to the ends of chromosomes (telomeres) to prevent loss of important genetic information due to the end-replication problem. Telomerase contains an RNA template and acts as a reverse transcriptase. 7. Proofreading and Error Correction Proofreading: DNA polymerases have proofreading activity (3’ to 5’ exonuclease activity) that allows them to remove incorrectly paired nucleotides immediately after they are added. Mismatch Repair: After replication, mismatch repair enzymes correct any errors that escaped the proofreading mechanism. 8. Regulation of Replication Cell Cycle Control: The initiation of replication is tightly regulated by the cell cycle. CDKs and other cell cycle regulators ensure that replication occurs only once per cell cycle. Checkpoints: DNA damage checkpoints, such as the G1/S and G2/M checkpoints, monitor the integrity of the DNA before and after
- 3. replication. If DNA damage is detected, the replication process can be paused to allow repair. Key Enzymes and Proteins Origin Recognition Complex (ORC) Cdc6 and Cdt1 MCM Helicase DNA Polymerases α, δ, and ε Primase RPA (Replication Protein A) RNase H FEN1 (Flap Endonuclease 1) DNA Ligase Telomerase Important Features of Eukaryotic DNA Replication Multiple Origins of Replication: Eukaryotic chromosomes have multiple origins of replication to ensure that the entire genome is replicated efficiently. Linear Chromosomes: Eukaryotic chromosomes are linear, unlike the circular chromosomes of prokaryotes, leading to specific challenges at the chromosome ends (telomeres). Replication Licensing: Eukaryotic cells ensure that replication occurs only once per cell cycle by tightly regulating the assembly of the pre- replication complex. This process ensures that eukaryotic cells accurately duplicate their DNA in preparation for cell division.