DNA Replication PDF

Replication of DNA Principles of Genetics, 6th Ed., by Snustad & Simmons, 2012, Ch. 10, pp. 220-246 How Does the DNA Replicate Itself and Transmit Genetic Information to a Daughter Cell? 1958: Meselson and Stahl Experiment The three possible modes of DNA replication Semiconservativ...

Replication of DNA Principles of Genetics, 6th Ed., by Snustad & Simmons, 2012, Ch. 10, pp. 220-246 How Does the DNA Replicate Itself and Transmit Genetic Information to a Daughter Cell? 1958: Meselson and Stahl Experiment The three possible modes of DNA replication Semiconservative DNA replicatio Watson and Crick first proposed this mechanism of DNA replication based on complementary basepairing between the two strands of the double helix. Note that each of the parental strands is conserved and serves as a template for the synthesis of a new complementary strand; that is, the base sequence in each progeny strand is determined by the hydrogenbonding potentials of the bases in the parental strand CsCl equilibrium density- gradient centrifugation CsCl equilibrium density- gradient centrifugation (Cont.) CsCl equilibrium density- gradient centrifugation (Cont.) Meselson and Stahl’s demonstration of semiconservative DNA replication in E. coli Meselson–Stahl experiment UNIQUE ORIGINS OF REPLICATION Visualization of the replication of the E. coli chromosome by autoradiography Bidirectional replication of the circular E. coli chromosome The use of AT-rich denaturation sites as physical markers to prove that the phage lambda chromosome replicates bidirectionally rather than unidirectionally Mechanism of action of DNA polymerases: covalent extension of a DNA primer strand in the 5’ → 3’ direction Evidence for discontinuous synthesis of the lagging strand DNA ligase catalyzes the covalent closure of nicks in DNA  The initiation of DNA strands with RNA primers The E. coli DNA primase is the product of the dnaG gene. In prokaryotes, these RNA primers are 10 to 60 nucleotides long, whereas in eukaryotes they are shorter, only about 10 nucleotides long The three activities of DNA polymerase I in E. coli Synthesis and replacement of RNA primers during replication of the lagging strand of DNA Proofreading by the 3’ → 5’ exonuclease activity of DNA polymerases during DNA replication. As introduced in Figure, the DNA molecules are shown as “stick” diagrams. If DNA polymerase is presented with a template and primer containing a 3 primer terminal mismatch (a), it will not catalyze covalent extension (polymerization). Instead, the 3’→ 5’ exonuclease activity, an integral part of many DNA polymerases, will cleave off the mismatched terminal nucleotide (b). Then, presented with a correctly base-paired primer terminus, DNA polymerase will catalyze 5’ → 3’ covalent extension of the primer strand (c).  Diagram of the E. coli replisome, showing the two catalytic cores of DNA polymerase III replicating the leading and lagging strands and the primosome unwinding the parental double helix and initiating the synthesis of new chains with RNA primers Evidence for bidirectional replication of the multiple replicons in the giant DNA molecules of eukaryotes The giant DNA molecules in the largest chromosomes of Drosophila melanogaster contain about 6.5 107 nucleotide pairs. The rate of DNA replication in Drosophila is about 2600 nucleotide pairs per minute at 25C. A single replication fork would therefore take about 17.5 days to replicate one of these giant DNA molecules. With two replication forks moving bidirectionally from a central origin, such a DNA molecule could be replicated in just over 8.5 days. Given that the chromosomes of Drosophila embryos replicate within 3 to 4 minutes and the nuclei divide once every 9 to 10 minutes during the early cleavage divisions, it is clear that each giant DNA molecule must contain many origins of replication. Indeed, the complete replication of the DNA of the largest Drosophila chromosome within 3.5 minutes would require over 7000 replication forks distributed at equal intervals along the molecule Thank You