1. 1 Review of directionality in DNA Now, for DNA replication.

2. 2 Meselson and Stahl: DNA replication is semiconservative Theoretically, 3 ways a DNA molecule could give rise to 2 new DNA molecules: Semi-conservative means that each time DNA is replicated, the new double stranded molecules consist of one old strand and one new strand. Conservative would result in a molecule with 2 old strands and one with 2 new ones. Dispersed, each new DNA molecule would be a combination of old and new pieces.

3. 3 Illustration of 3 models http://en.wikipedia.org/wiki/Image:DNAreplicationModes.png

4. 4 Meselson and Stahl Experiment DNA was produced in cells grown with N-15, a “heavy” isotope of nitrogen. When DNA was placed into an ultracentrifuge, it migrated closer to the bottom because of its greater density. What happened when N-15 labeled cells were allowed to keep growing in the presence of N-14? http://users.rcn.com/jkimball.ma. ultranet/BiologyPages/M/Mesels on_Stahl.html

5. 5 Conclusion of experiment After 1 generation, all the DNA molecules of intermediate density. After 2 generations, half of them intermediate, the other half light. These results consistent with semi-conservative replication. http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/M/Meselson_Stahl.html

6. 6 http://www.sciencetechnologyaction.com/lessons2.php?s tudyid=6&edition=1 “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material." -Watson and Crick

7. 7 DNA replication Origin of DNA replication: particular site on DNA where copying of the DNA always starts. –Replication is bidirectional –In each direction, there is a replication fork. –Most bacterial DNA is circular, so there is one Origin and one terminus Replicon: a length of DNA molecule replicated after initiation from one origin. Examples: –Bacterial DNA, plasmids, segments of eukaryotic chromosomes.

8. 8 DNA replication terminology In linear DNA, origins occur at various places within the DNA molecule. The DNA replicated from one origin is a replicon. In circular bacterial genomes, replication proceeds from a single origin; the entire molecule is a replicon.

9. How long does it take? 9 DNA replication is a complicated process involving a variety of enzymes and other proteins, so it takes a while. Speed of replication: –Bacteria: 1500 bp per second –Eukaryotes: 10-100 bp per sec.YET in fruit flies, only 15-30 minutes to replicate all the DNA, similar to E. coli. How? Multiple origins.

10. 10 E. coli, a typical impatient bacterium E. coli takes 30 minutes to replicate all its DNA, yet it can double every 20 minutes. How does it do this? Starts a round of DNA replication before finishing the previous round.

11. 11 DNA polymerases (bacteria): Enzymes that synthesize DNA Kornberg discovers DNA pol I (1956) –Demonstrates enzyme faithfully copies DNA (1960) DNA pol II and III discovered –Pol I: cleaves out Okazaki fragments (see below) Most abundant of the 3. –Pol II: repairs DNA damage –Pol III: main DNA replicating enzyme Pol III is a complex, multi-component enzyme complex (has a quaternary structure)

12. 12 A couple of words on terminology A chemical reaction in which molecules are combined to make a products is a synthesis reaction. DNA is synthesized in cells, but we can direct DNA synthesis in a test tube also. PCR, sequencing both involve DNA synthesis. DNA replication is a natural biological process in which a DNA molecule is copied in a cell. –Replication is a specific act of synthesis.

13. 13 What every DNA polymerase needs A template of DNA –Enzymes copy a single strand of DNA –Can’t work without something to copy from A primer –A primer is a polynucleotide with a “free 3´OH end” –In normal DNA replication, this is RNA A substrate –To make DNA, a polymer, monomers are needed –Nucleotide triphosphates (NTPs) are the monomers

14. Numbering of ring positions 14 Ring positions on nitrogenous bases “use up” the numbers, so positions on sugar are indicated by “prime”. 5’ and 3’ positions on sugar are very important.

15. 15 Adding and removing bases: Directionality DNA synthesis is ALWAYS in a 5´ to 3´ direction –See next slide. All 3 DNA pols have a 3´ to 5´ exonuclease activity –Nuclease: enzyme activity that cuts nucleic acids –Exo- means cuts from an end –3´ to 5´ means the opposite direction from synthesis “proofreading” ability; polymerase can “backspace” to remove a base put it by mistake. DNA pol I has a 5´ to 3´ exonuclease activity –Cuts off DNA bases in same direction as synthesis

16. 16 * * * *

17. 17 Initiation of DNA replication DNA pol requires a primer to add to: Primase makes an RNA Synthesis is 5´ to 3´, and antiparallel. Leading strand; synthesis follows replication fork. * Helicases unwind the DNA *

18. 18 Problems due to antiparallel nature of DNA In this picture, replication of the lower strand of DNA can proceed as the “replication fork” moves from right to left because the direction of synthesis of new DNA is 5’ to 3’. What about the other strand? The one made without a hitch is called the “leading strand”, the other is the “lagging strand”.

19. 19 Okazaki fragments Because of requirement for 5’to 3’ synthesis, lagging strand must repeatedly top and start; needs an RNA primer each time.

20. 20 Cleaning up Okazaki’s DNA Pol I cuts out RNA primers, replaces them with DNA. Uses both the 5´ to 3´ exonuclease and polymerase activities. Ligase needed

21. The need for Ligase 21 A new nucleoside triphosphate could be added onto the 3’ end, but there is no way a polymerase can join a 3’ end to an existing 5’ end of a DNA strand that has a single phosphate group. This is the job of the enzyme ligase. It makes a covalent bond to connect ends after Okazaki fragments are replaced and is used as a tool in recombinant DNA technology.

22. 22 Facts about eukaryotic DNA synthesis Multiple replicons –Makes up for slower synthesis & larger amounts of DNA 6 polymerases –Greek letters instead of Roman numerals: Pol α – ε –Pol α creates RNA primer, adds some DNA, falls off “low processivity” –Pol δ takes over Faster, and has proofreading ability Pol ε does same thing under different conditions