(15) DNA replication

(15) DNA replication - DNA Replication Zilong Wen...

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± Zilong Wen Department of Biochemistry ([email protected]; Room 5522) DNA Replication
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± Cell Cycle
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± The Key Event: DNA Replication
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± Basic Rules of DNA Replication • Semi-conservative replication • DNA synthesis cannot start de novo , but requires templates and primers • DNA synthesis proceeds in a 5’ 3’ direction and is semi-discontinuous • DNA replication starts at an Origin and usually proceeds bi-directionally in replication forks
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± Escherichia coli ( E. Coli ) Bacterium A model organism used in the study of genetics, physiology, and biochemistry.
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± Basic Rules of DNA Replication • Semi-conservative replication • DNA synthesis cannot start de novo , but requires a templates, primers and proteins • DNA synthesis proceeds in a 5’ 3’ direction and is template-dependent • DNA replication starts at an Origin and usually proceeds bi-directionally in replication forks
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± Semi-conservative Conservative Dispersive Semi-conservative DNA Replication James Watson and Francis Crick Matthew Meselson & Franklin Stahl in 1958
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± Ultracentrifugation to separate molecules based on their densities Load sample on top of dense (sucrose, CsCl) solution Spin to separate different molecules Meselson-Stahl’s Experiment Meselson-Stahl Experiment: Different molecules have different sedimentation coefficients when subjected to ultracentrifugation.
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± Meselson-Stahl’s Experiment In alkaline condition, double- stranded DNA is denatured into single strands. DNA molecules can migrate as a single band and species with different densities can be separated. DNA containing 15 N has a higher density than that with 14 N.
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±² Meselson-Stahl’s Experiment The experiment The interpretation
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±± Density Gradient Under Different pH Relative DNA concentration 15 N 14 N DNA from cells grown in 15 N, then one generation in 14 N 15 N, then two generations in 14 N pH=7.0 (intact DNA) pH=12.0 (denatured) Density, g/cc 1.71 1.717 1.724 1.76 1.774
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±² Semi-conservative DNA Replication
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±² Basic Rules of DNA Replication • Semi-conservative replication • DNA synthesis cannot start de novo , but requires templates and primers • DNA synthesis proceeds in a 5’ 3’ direction and is semi-discontinuous • DNA replication starts at an Origin and usually proceeds bi-directionally in replication forks
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