DNA Replication Lecture Notes PDF

DNA replication DNA Replication In prokaryotes, replication occurs in the cytoplasm, while in the eukaryotes, replication takes place in the nucleus. Replication is bidirectional from the origin (starting point) for prokaryotes & eukaryotes. Watson and Crick DNA model implies a mechanism for replica...

DNA replication DNA Replication In prokaryotes, replication occurs in the cytoplasm, while in the eukaryotes, replication takes place in the nucleus. Replication is bidirectional from the origin (starting point) for prokaryotes & eukaryotes. Watson and Crick DNA model implies a mechanism for replication: a. Unwind the DNA molecule. b. Separate the 2 strands. c. Make a complementary copy for each strand. 3 possible models were proposed for DNA replication: ✓ Conservative model ✓ Dispersive model ✓ Semiconservative model 22 NOV 2023 MIBS BATCH 29 5 of DNA 3 proposed models for the replication of DNA DNA strands separate and a complementary strand is synthesized for each, so that sibling chromatids have one old and one new strand. 22 NOV 2023 both strands of one copy would be entirely old DNA, while the other copy would have both strands of new DNA. MIBS BATCH 29 dsDNA might fragment, replicate dsDNA, and then reassemble, creating a mosaic of old and new dsDNA regions in each new chromosome. 6 The Matthew Meselson & Frank Stahl experiment Step 1: E. coli was cultured in a 15N medium. DNA was isolated. Step 2: The culture was shifted to a 14N medium & grown. Step 3: DNA was isolated at different times corresponding to replication cycles 0, 1, 2 & 3. Nitrogen is a major constituent of DNA. 14N is by far the most abundant isotope of nitrogen, but DNA with the heavier (but non-radioactive) 15N isotope is also functional. Step 4: The extracted DNA samples were centrifuged on a salt (CsCl) density gradient. DNA separates out at the point at which its density equals that of the salt solution. 22 NOV 2023 MIBS BATCH 29 7 The Matthew Meselson & Frank Stahl experiment Results 1) After 1 replication cycle in a 15N containing medium (cycle 0), the DNA synthesized had a higher density. 15N is a heavy isotope of nitrogen. 2) After 1 replication cycle in a 14N medium (cycle 1), the DNA was all of intermediate density. 3) After 2 replication cycles in a 14N medium (cycle 2) , there were 2 bands, 1 of intermediate density and 1 of the lighter density corresponding with 14N. 4) After 3 cycles in a 14N medium, 2 bands were observed, 1 light & 1 intermediate form decreasing over time. 22 NOV 2023 MIBS BATCH 29 8 The Meselson-Stahl experiment: showed that DNA replicates semi-conservatively Interpretation Results compared with the 3 proposed models: a. Does not fit conservative model because after one generation there is a single intermediate band, rather than one with entirely 15N DNA and another with entirely 14N DNA. b. The dispersive model predicted that a single band of DNA of intermediate density would be present in each generation, gradually becoming less dense as increasing amounts of 14N were incorporated with each round of replication. Instead, Meselson and Stahl observed 2 bands of DNA (intermediate & light in the 3rd cycle), with the intermediate form decreasing over time. c. The semiconservative model fits the data very well. 22 NOV 2023 MIBS BATCH 29 9