f3 - DNA Replication Semi-conservative nature Prokaryotic...

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• Prokaryotic DNA Replication • Eukaryotic DNA Replication Readings: Ch. 4: pp 131-136; Ch. 10: pp 435-437 DNA Replication: Semi-conservative nature
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DNA Replication Occurs During S- Phase of Cell Division S phase (DNA synthesis; chromosome duplication) Interphase (90% of time) G 1 Mitotic phase (M) (10% of time) Cytokinesis Mitosis G 2 DNA Replication (S phase) Cell Division (Mitosis/cytokinesis)
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Parental DNA First-generation Progeny DNA Second-generation progeny Hypothesis 1 Hypothesis 2 Hypothesis 3 What is the nature of DNA Replication?
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Use density gradient centrifugation to separate DNA to analyze the change of density of DNA isolated. Nature of DNA Replication: Experiment by Meselson & Stahl
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Hypothetical Result: Nature of DNA Replication: Experiment by Meselson & Stahl
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Experimental Result: Nature of DNA Replication: Experiment by Meselson & Stahl • Before N 14 transfer (0), only HH DNA • ~1 generation (0.7-1.5), only HL DNA • ~2 generations (1.9- 3.0), both HL and LL DNA • ~4 generations after, only LL DNA
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Newly synthesized daughter strand Conserved parental strand DNA Replication in Eukaryotic Cells
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Origin of Replication Where will replication start in a genome? Replication origin (or Ori , replicon ) is a specific sequence in the genome, in which the dsDNA is first opened up for replication. Prokaryotes — Most prokaryotes have one replicon per circular genome (see left). Plasmid and viruses also have only one replicon per genome. But prokaryotic sequencing projects have revealed that some prokaryotes (such as Agrobacteria) have linear chromosome and multiple replicons.
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• The genome of eukaryotes are much larger than prokaryotes. Takes too long to completely replicate entire genome in a cell with one origin of replication. • Issue is resolved if have multiple origins of replication… 10,000-100,000 replicons A eukaryotic genome has many origins of replication per chromosome
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Bacterial OriC Features of Replication Origins 1. Core promoter DNA sequences are short repeats that are rich in A/T . –E. coli OriC is ~248 bp; one core sequence is TGTGGATAA –Yeast have ARS1 (autonomous replication sequence 1); core sequence: A/TTTTAA/GTTTA/T 2. They are bound by specific ori-binding proteins regardless of replication (e.g. Dna A in E. coli or ORC in SV40). The Origin-binding proteins will bind to additional enzymes in order to start replication.
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Unidirectional vs Bidirectional Replication • DNA synthesis is directional: always from 5’ 3’ • Problem: a dsDNA contains two anti-parallel ssDNA strands, but the DNA synthesis can only be 5’ to 3’. • There are three possible solutions, by which a dsDNA can be synthesized. What actually happens in the cell?
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Bidirectional Replication • SV40 viral circular DNA was cut near the origin of replication by an enzyme, EcoR I • The replication bubble gets increasingly longer from both direction…the center is constant (note change in distance from ends of DNA) • Replication is bidirectional
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Implications of bi-directional replication: One replication bubble has two forks growing in the opposite direction.
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