免疫学信息网 http:/www.immuneweb.com,Electron Microscopy of replicating DNA reveals replicating bubbles. How does one prove bidirectional fork movement?,免疫学信息网 http:/www.immuneweb.com,Pulse with radiolabeled nucleotide; chase with cold nucleotide. Then do autoradiography,免疫学信息网 http:/www.immuneweb.com,DNA Replication DNA replication is semi-conservative, one strand serves as the template for the second strand. Furthermore, DNA replication only occurs at a specific step in the cell cycle. The following table describes the cell cycle for a hypothetical cell with a 24 hr cycle. Stage Activity Duration G1 Growth and increase in cell size 10 hr S DNA synthesis 8 hr G2 Post-DNA synthesis 5 hr M Mitosis 1 hr DNA replication has two requirements that must be met: 1. DNA template 2. Free 3 -OH group,免疫学信息网 http:/www.immuneweb.com,免疫学信息网 http:/www.immuneweb.com,DNA Replication DNA replication is semi-conservative, one strand serves as the template for the second strand. Furthermore, DNA replication only occurs at a specific step in the cell cycle. The following table describes the cell cycle for a hypothetical cell with a 24 hr cycle. Stage Activity Duration G1 Growth and increase in cell size 10 hr S DNA synthesis 8 hr G2 Post-DNA synthesis 5 hr M Mitosis 1 hr DNA replication has two requirements that must be met: 1. DNA template 2. Free 3 -OH group,免疫学信息网 http:/www.immuneweb.com,免疫学信息网 http:/www.immuneweb.com,Proteins of DNA Replication DNA exists in the nucleus as a condensed, compact structure. To prepare DNA for replication, a series of proteins aid in the unwinding and separation of the double-stranded DNA molecule. These proteins are required because DNA must be single-stranded before replication can proceed. 1. DNA Helicases - These proteins bind to the double stranded DNA and stimulate the separation of the two strands. 2. DNA single-stranded binding proteins - These proteins bind to the DNA as a tetramer and stabilize the single-stranded structure that is generated by the action of the helicases. Replication is 100 times faster when these proteins are attached to the single- stranded DNA. 3. DNA Topoisomerase - This enzyme catalyzes the formation of negative supercoils that is thought to aid with the unwinding process. In addition to these proteins, several other enzymes are involved in bacterial DNA replication.,免疫学信息网 http:/www.immuneweb.com,4. DNA Polymerase - DNA Polymerase I (Pol I) was the first enzyme discovered with polymerase activity, and it is the best characterized enzyme. Although this was the first enzyme to be discovered that had the required polymerase activities, it is not the primary enzyme involved with bacterial DNA replication. That enzyme is DNA Polymerase III (Pol III). Three activities are associated with DNA polymerase I; * 5 to 3 elongation (polymerase activity) * 3 to 5 exonuclease (proof-reading activity) * 5 to 3 exonuclease (repair activity) The second two activities of DNA Pol I are important for replication, but DNA Polymerase III (Pol III) is the enzyme that performs the 5-3 polymerase function. 5. Primase - The requirement for a free 3 hydroxyl group is fulfilled by the RNA primers that are synthesized at the initiation sites by these enzymes. 6. DNA Ligase - Nicks occur in the developing molecule because the RNA primer is removed and synthesis proceeds in a discontinuous manner on the lagging strand. The final replication product does not have any nicks because DNA ligase forms a covalent phosphodiester linkage between 3-hydroxyl and 5-phosphate groups.,免疫学信息网 http:/www.immuneweb.com,A General Model for DNA Replication 1. The DNA molecule is unwound and prepared for synthesis by the action of DNA gyrase, DNA helicase and the single-stranded DNA binding proteins. 2. A free 3OH group is required for replication, but when the two chains separate no group of that nature exists. RNA primers are synthesized, and the free 3OH of the primer is used to begin replication. 3. The replication fork moves in one direction, but DNA replication only goes in the 5 to 3 direction. This paradox is resolved by the use of Okazaki fragments. These are short, discontinuous replication products that are produced off the lagging strand. This is in comparison to the continuous strand that is made off the leading strand. 4. The final product does not have RNA stretches in it. These are removed by the 5 to 3 exonuclease action of Polymerase I. 5. The final product does not have any gaps in the DNA that result from the removal of the RNA primer. These are filled in by the 5 to 3 polymerase action of DNA Polymerase I. 6. DNA polymerase does not have the ability to form the final bond. This is done by the enzyme DNA ligase.,免疫学信息网 http:/www.immuneweb.com,RNA primed DNA replication,免疫学信息网 http:/www.immuneweb.com,A General Model for DNA Replication 1. The DNA molecule is unwound and prepared for synthesis by the action of DNA gyrase, DNA helicase and the single-