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DNA replication (4 lectures) and cell-cycle (1 lecture) Anindya Dutta Presentations at the MICR811 toolkit site Also at http://www.cs.virginia.edu/~am3cp/DNARepl/ http://mexico.bioch.virginia.edu/DNARepl Electron Microscopy of replicating DNA reveals replicating bubbles. How does one prove bidirectional fork movement? Pulse with radiolabeled nucleotide; chase with cold nucleotide. Then do autoradiography 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 G1 S G2 M Activity Growth and increase in cell size DNA synthesis Post-DNA synthesis Mitosis Duration 10 hr 8 hr 5 hr 1 hr DNA replication has two requirements that must be met: 1. 2. DNA template Free 3' -OH group 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 G1 S G2 M Activity Growth and increase in cell size DNA synthesis Post-DNA synthesis Mitosis Duration 10 hr 8 hr 5 hr 1 hr DNA replication has two requirements that must be met: 1. 2. DNA template Free 3' -OH group 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 singlestranded 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. 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. 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 3'OH group is required for replication, but when the two chains separate no group of that nature exists. RNA primers are synthesized, and the free 3'OH 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. RNA primed DNA replication 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 3'OH group is required for replication, but when the two chains separate no group of that nature exists. RNA primers are synthesized, and the free 3'OH 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. 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 3'OH group is required for replication, but when the two chains separate no group of that nature exists. RNA primers are synthesized, and the free 3'OH 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. Removal of RNA primers and filling of gaps 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 3'OH group is required for replication, but when the two chains separate no group of that nature exists. RNA primers are synthesized, and the free 3'OH 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. ATP is an integral part of the ligation reaction The discovery of DNA polymerase. Arthur Kornberg and Bob Lehman pursued an enzyme in bacterial extracts that would elongate a chain of deoxyribonucleic acid just like glycogen synthase elongates a chain of glycogen. The enzymatic activity was unusual: 1) Needed a template which dictates what nucleotide was added: substrate was directing enzymatic activity 2) Needed a primer annealed to the template. exonuclease I Wait a minute! Either the polymerase hypothesis was all wrong,…… or there were other DNA polymerases in E. coli that carried out DNA synthesis in the polA strains. 200 polA + (wild type) 0.4M II III 0.2M 100 +NEM polA- (Cairns) 200 II 0.4M III 0.2M 100 I 20 30 Fractions 40 Phosphate (M) John Cairns mutated the gene for DNA polymerase, polA, and the bacteria grew just fine! 3H Thymidine incorporation (pmol) 600 Sub kDa unit Gene Subassembly 130 27.5 | | POL III CORE dnaE dnaQ (mutD) 10 71 dnaX ' 47.5 35 33 15 12 dnaX 40.6 dnaN | | | | COMPLEX | | CLAMP DNA POL YMERASE III 5'-3' polymerase 5'-3' exonuclease 3'-5' exonuclease ATP dependent clamploader processivity factor The end-replication problem Solution to the end-replication problem Telomerase Telomeres contain arrays of DNA repeats Group Organism Telomeric repeat (5' to 3' toward the end) Vertebrates Human, mouse, Xenopus TTAGGG Filamentous fungi Neurospora TTAGGG Physarum, Didymium Dictyostelium TTAGGG AG(1-8) Tetrahymena, Glaucoma Paramecium Oxytricha, Stylonychia, Euplotes TTGGGG TTGGG(T/G) TTTTGGGG Schizosaccharomyces pombe TTAC(A)(C)G(1-8) Slime molds Ciliated protozoa Fission yeasts Budding yeasts Saccharomyces cerevisiae TGTGGGTGTGGTG Telomerase is a reverse transcriptase together with a template RNA It is active in germ cells, not in somatic cells, and is activated in cancers Looping the lagging strand to make both polymerases move in the same direction Sub kDa unit Gene Subassembly 130 27.5 | | POL III CORE dnaE dnaQ (mutD) 10 71 dnaX ' 47.5 35 33 15 12 dnaX 40.6 dnaN | | | | COMPLEX | | CLAMP DNA POL YMERASE III 5'-3' polymerase 5'-3' exonuclease 3'-5' exonuclease ATP dependent clamploader processivity factor Sub Gene unit Bacterial Function Eukaryotic dnaE | DNA POL dnaQ (mutD) | POL III CORE | 5'-3' polymerase 3'-5' exonuclease 5'-3' exonuclease | | | COMPLEX | | ATP dependent clamploader dnaX ' dnaX dnaN CLAMP processivity factor DNA POL Fen1 RF-C PCNA CONSERVATION FROM PROKARYOTES TO EUKARYOTES Which polymerase is processive? P POL dNTP Challenge with vast excess of cold primer-template Gel electrophoresis of products Challenge - + POL-X - + POL-Y POLIII, subunit PCNA Clamp loaders hydrolyze ATP to load clamp Clamp-loader ATP ATP Clamp How does one prove that the clamp ring is opened during loading? ATP ADP + PP i 3‘OH Structure of a DNA polymerase (gp43 from phage RB69) Side view: Polymerase active site Top view with template-primer: Polymerase site And proofreading site Topoisomerases relax DNA by changing the DNA linking number * Topoisomerases II change the linking number in steps of 2 by passing both strands of double-stranded DNA through a break. * Eukaryotic topoisomerases isolated to date only relax supercoiled DNA, while prokaryotic topoisomerases (gyrases) can, given ATP, add supercoils. * TopoII releases catenated daughter molecules at the end of replication. Inhibitors like etoposide are used in chemotherapy. * Topoisomerases I change the linking number in steps of 1. They pass a single DNA strand through a nick.Topoisomerase I is a protein of the metaphase chromosome scaffold. * In interphase, topoisomerase is bound to the nuclear matrix. * The DNA replication machinery also appears bound to the matrix. * Inhibitor (camptothecin) also used in chemotherapy. Topoisomerase action can be divided into three steps: nicking (1), strand passage (2); resealing (3). 5‘ end of DNA in gate segment is covalently linked to the OH of tyrosine in the active site of topo. Cycle of topoisomerase activity inferred from structure 1 2 4 3 How would you test that the subunits have to open at the lower end to release the T segment?