The Flow of Genetic Information. MBLG1001 Lecture 9. Replication. Is the process : The Messelson Stahl Experiment. The Messelson Stahl Experiment

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1 The Flow of Genetic Information MBLG1001 Lecture 9 Replication Chapter 7 Malacinski Chapter 5 Clark Transcription Translation DNA RNA rotein replication DNA Folding, modification, translocation Functional protein Figure 28.1 Watson and Crick s famous paper, in its entirety. (Reprinted with permission from Watson,J.D., and Crick,F..C., Molecular structure of nucleic acid, Nature 171: Copyright 1953 Macmillan ublishers Ltd.) Replication Is the process : Conservative, Semi-conservative R Dispersive? The Messelson Stahl Experiment Cells were grown up on the heavy isotope of N, 15 N, abbreviated as ( 15 N 4 Cl) then the medium was changed to one containing normal 14 N (light or L) as sole nitrogen source. DNA was isolated at various time points after the media change and applied to a CsCl density gradient. The Messelson Stahl Experiment This technique separates by buoyant density DNA containing 2 light strands (L:L) will sediment at a different density to a hybrid eavy:light (:L) nucleic acid or the eavy:eavy (:) form of DNA. ld parent DNA will be heavy () Newly synthesised DNA will be light (L). 1

2 The Messelson Stahl Experiment If replication was conservative LL L Concentrated CsCl solutions, when centrifuged really fast form a gradient. Compounds separate by their buoyant density in such a gradient. The DNA one cell division after medium change would be composed of : and L:L in equal proportions. In the second generation there should be 3 L:L to 1 :. The third generation..7 L:L and 1 : If replication was semi conservative In the first generation after medium change the DNA would be composed of solely :L In the next generation you would expect :L and L:L in a ratio of 1:1. In the following generation the :L and L:L would have a ratio of 1:3. In the next generation it would be 1:7. If replication was dispersive ybrid :L DNA would result but if the individual strands were analysed under denaturing conditions (in CsCl with Na to keep the strands apart) they would also have an intermediate density. The individual DNA strands would always be completely or L in the other models. E. coli Replication: the quintessential example E. coli can, under optimal growth conditions double cell numbers every 20 min. Clark p125 It has 1 large circular chromosome; 4.6 million bp The replication fork moves at a constant 1000 NMs/sec. There are 2 forks which move in opposite directions 2

3 E. coli Replication: the quintessential example At this rate it takes 40 min to copy the whole E. coli genome (4.6 million bases pairs) and another 20 min to separate the cellular components. To double in less than 60 min means the cell must initiate the next round of replication before the previous one had finished. To scale up this process it is a 400 k trip made by 2 machines in 40 min with an error made every 170 k. oric The replication forks E. coli s problems: Replication is bi-directional. The theta model. Bacterial DNA is a closed circle so it will get tangled when it is unwound. Enzymes are needed to copy the DNA The strands must be pulled apart and unwound. What enzymes are involved in copying DNA? As soon as the structure of DNA was elucidated the hunt was on for the enzymes which copy it. These enzymes are known as polymerases ver the past 50 years many such enzymes have been found. Some even copy an RNA. The discovery of DNA polymerases Arthur Kornberg The first DNA polymerase (DNA pol I) was isolated in 1956, only 3 years after the structure of DNA was published. Arthur Kornberg isolated DNA pol I and won the 1959 Nobel prize for his efforts. At the time it was thought to be the main replicative enzyme. 3

4 roblems with DNA pol I It didn t work fast enough to copy the whole genome. John Cairns and aula DeLucia isolated mutants of E. coli which had ~1% of the DNA pol I activity but still divided at normal rates. Moral of the story: get the prize before anyone can prove you wrong. Search for DNA copying enzymes Since then another 4 polymerases have been identified in E. coli: DNA pol II, III, IV and V These have been isolated and purified from pola- mutants. DNA pols II, IV and V are repair enzymes and DNA pol III was the Big one! There have been to date over 15 eukaryotic DNA pols isolated and purified and some interesting viral versions. DNA polymerase III The father and son act! It wasn t until 1970 that DNA pol III was isolated by Arthur s son, Thomas. This is a truly large enzyme with ~10 subunits. It has a circular donut-like pair of subunits which clamp the enzyme to the DNA. This gives it its processivity (ability to remain tightly associated with the template through many nucleotide additions) The main players in E coli Replication. DNA polymerase I has : 5 to 3 exonuclease 3 to 5 exonuclease (proof reading) 5 to 3 polymerase (new strand) DNA polymerase III has; 3 to 5 exonuclease (proof reading) 5 to 3 polymerase (new strand) roperties of DNA polymerases Clark p187, Malacinski p to 3 polymerase activity; All polymerases (DNA and RNA) synthesise the new strand of nucleic acid in a 5 to 3 direction. All DNA polymerases need a primer; a short fragment of single stranded nucleic acid bound to the template which provides a 3 to make the next addition. All DNA polymerases have a 3 to 5 exonuclease activity 4

5 5 to 3 polymerase activity arent strand to 3 polymerase activity arent strand 5 3 dnt 5 dnt 5 The correct dnt which base pairs to the template base is added by the polymerase The correct dnt which base pairs to the template base is added by the polymerase 5 to 3 polymerase activity arent strand to 3 polymerase activity arent strand 5 3 dnt 5 3 Newly synthesised strand 5 The correct dnt which base pairs to the template base is added by the polymerase Eventually the whole strand is copied Growing strand (n) residues long At the molecular level - - : Growing strand now (n+1) residues long Cleaving these phosphodiester bonds provides the energy for the polymerisation - C 2 New incoming nucleotide triphosphate: dnt yrophosphate i

6 Rapidly breaks down to 2 phosphates Growing strand now (n+1) residues long roof reading or editing Clark p 113 Malcinski p 129 DNA polymerases, and not RNA polymerases, have an editing function. The 3 to 5 exonuclease is a slow acting nuclease It cleaves the newly added nucleotide if it does not base pair properly to the template. yrophosphate i The rimer Clark p115 Malacinski p 135 All DNA polymerases need a primer, even reverse transcriptase and Klenow. RNA polymerases do NT need a primer. They generate the primer for DNA synthesis. The need for a 3 is exploited in drug design and certain techniques e.g. DNA sequencing. A quick journey through the other polymerases Klenow enzyme or fragment. This enzyme comes from DNA pol I. If you digest DNA pol I for a short amount of time with a protease (called limited proteolysis) you get 2 fragments: A ~66 kd fragment with polymerase and 3 to 5 exonuclease activity. A~33 kd fragment with 5 to 3 exonuclease activity. Klenow enzyme or fragment. The larger fragment is the Klenow enzyme. It is very useful as a DNA polymerase. It requires a primer (needs a 3 to add the next nucleotide to). It is very good a copying DNA. It can be used to synthesise a labeled strand of DNA for experiments 6

7 The history of DNA olymerases Reverse Transcriptase roduced by retroviruses e.g. IV Uses an RNA template roduces a DNA copy, known as complementary DNA or cdna Works 5 to 3 and requires a primer. First isolated in 1970 by oward Temin and David Baltimore independently. Figure The structures of AZT (3 -azido-2,3 - dideoxythymidine). This nucleoside was the first approved drug for treatment of AIDS. AZT is phosphorylated in vivo to give AZTT (AZT 5 -triphosphate), a substrate analog that binds to IV reverse transcriptase, IV reverse transcriptase incorporates AZTT into growing DNA chains in place of dtt. Incorporated AZTM blocks further chain elongation because its 3 -azido group cannot form a phosphodiester bond with an incoming nucleotide. ost cell DNA polymerases have little affinity for AZTT. Taq olymerase A thermal stable DNA polymerase isolated form the bacterium, Thermus aquaticus which lives in the hot springs of Yellowstone National ark. Used in a reaction known as olymerase Chain Reaction (CR). CR is able to amplify sections of DNA by copying it over and over. ther DNA polymerases Repair: DNA pol IV and pol V. Eukaryotic DNA polymerases: α, β, δ, and ε with γ found in mitochondria. Then there are the eukaryotic repair enzymes!! ALL WRK to make a new strand in the 5 to 3 orientation. So what do we know there are a lot of DNA polymerases which are capable of copying a strand of DNA, provided they are supplied with nucleotides, template and a primer. But how and when does replication occur? 7