DNA replication is a crucial process for life, copying genetic material before cell division. Prokaryotes and eukaryotes share basic principles but differ in complexity and regulation.
Prokaryotes have simpler, faster replication with one origin on a circular chromosome. Eukaryotes have multiple origins on linear chromosomes, deal with chromatin, and have stricter cell cycle control.
DNA Replication in Prokaryotes vs Eukaryotes
Fundamental Similarities and Differences
- Semi-conservative model applies to both prokaryotic and eukaryotic DNA replication generates new double helix with one original strand and one newly synthesized strand
- Prokaryotic DNA replication occurs in cytoplasm while eukaryotic DNA replication takes place in nucleus
- Prokaryotes typically have single circular chromosome with one origin of replication
- Eukaryotes possess multiple linear chromosomes with multiple origins of replication
- DNA replication speed in prokaryotes reaches approximately 1000 nucleotides/second
- Eukaryotic DNA replication proceeds at slower rate of approximately 50 nucleotides/second
- Eukaryotic DNA replication involves additional complexities
- Chromatin structure management
- Presence of telomeres at chromosome ends
- Prokaryotic DNA replication couples with cell division
- Eukaryotic DNA replication restricts to S phase of cell cycle
Structural and Functional Complexities
- Eukaryotic chromosomes contain histone proteins forming nucleosomes
- Nucleosomes must be disassembled and reassembled during replication
- Eukaryotic telomeres require specialized mechanisms for replication
- Telomerase enzyme adds repetitive sequences to chromosome ends
- Prokaryotic circular chromosome forms supercoiled structure
- Topoisomerases relieve DNA tension during replication
- Eukaryotic replication involves larger number of proteins and regulatory factors
- Minichromosome maintenance (MCM) proteins act as replicative helicases
- Proliferating cell nuclear antigen (PCNA) serves as sliding clamp for DNA polymerases
Origin of Replication in DNA Synthesis
Structure and Recognition of Origins
- Origin of replication (ori) consists of specific DNA sequence initiating replication
- Prokaryotic origin recognition
- DnaA protein binds to specific sequences called DnaA boxes
- DnaA boxes typically contain 9-base pair consensus sequence (TTATCCACA)
- Eukaryotic origin recognition
- Origin Recognition Complex (ORC) binds to replication origins
- ORC composed of six subunits (ORC1-6)
- Eukaryotic origins often associated with AT-rich sequences
- Binding of initiator proteins to origin leads to local unwinding of DNA double helix
- Replication bubble forms at unwound region
- Multiple proteins recruited to origin form pre-replication complex (pre-RC)
- Prokaryotes: DnaB helicase loaded onto DNA by DnaC protein
- Eukaryotes: MCM2-7 helicase complex loaded by Cdc6 and Cdt1 proteins
- Helicases further unwind DNA preparing template for replication
- Number and distribution of origins affect overall replication timing and efficiency
- Prokaryotes typically have single origin (oriC in E. coli)
- Eukaryotes contain thousands of origins spaced along chromosomes
Significance of the Replication Fork
Structure and Components of the Replication Fork
- Replication fork forms Y-shaped region where DNA strands separate for new strand synthesis
- Key components of replication fork
- DNA helicase unwinds parental DNA strands
- DNA primase synthesizes short RNA primers (5-10 nucleotides long)
- DNA polymerase initiates synthesis from 3' hydroxyl group of primers
- Single-stranded binding proteins (SSB) stabilize unwound single-stranded DNA
- Leading strand synthesized continuously in 5' to 3' direction
- Lagging strand synthesized discontinuously as Okazaki fragments
- Okazaki fragments typically 100-200 nucleotides long in prokaryotes
- Eukaryotic Okazaki fragments shorter (approximately 100-150 nucleotides)
Enzymatic Activities at the Replication Fork
- DNA polymerase III (prokaryotes) or DNA polymerase δ and ε (eukaryotes) catalyze nucleotide addition
- DNA ligase joins Okazaki fragments on lagging strand
- RNase H removes RNA primers
- DNA polymerase I (prokaryotes) or DNA polymerase δ (eukaryotes) fills gaps left by primer removal
- Topoisomerases relieve DNA supercoiling ahead of replication fork
- Prokaryotes use DNA gyrase (type II topoisomerase)
- Eukaryotes employ topoisomerase I and II
Regulation of DNA Replication: Prokaryotes vs Eukaryotes
Prokaryotic Replication Regulation
- Prokaryotic replication primarily regulated at initiation stage
- DnaA protein availability and activity control replication initiation
- DnaA-ATP active form binds origin, while DnaA-ADP inactive
- Regulatory mechanisms in prokaryotes
- Sequestration of newly replicated origins by SeqA protein (E. coli)
- Titration of DnaA protein by multiple DnaA binding sites across genome
- Regulation of DnaA gene expression
- Prokaryotes adjust replication rate in response to nutrient availability
- Nutrient-rich conditions increase initiation frequency
Eukaryotic Replication Regulation
- Eukaryotic replication tightly controlled throughout cell cycle
- Two-step process for origin activation
- Licensing of origins in G1 phase
- Activation of licensed origins in S phase
- Cyclin-dependent kinases (CDKs) play crucial role in regulating replication
- CDK activity low in G1 allows origin licensing
- CDK activity high in S phase promotes origin firing and prevents re-licensing
- Multiple mechanisms prevent re-replication within single cell cycle
- Degradation of licensing factors (Cdt1)
- Nuclear export of MCM proteins
- Inactivation of origins after firing
- Replication timing regulation in eukaryotes
- Concept of early and late-replicating domains
- Influenced by chromatin structure and nuclear organization
- Transcriptionally active regions often replicate early