1. DNA Replication

2. II- DNA Replication II- DNA Replication Origins of replication Origins of replication Replication ForkshundredsY-shaped replicating DNA molecules 1.Replication Forks: hundreds of Y-shaped regions of replicating DNA molecules where new strands are growing. ReplicationFork Parental DNA Molecule 3’ 5’ 3’ 5’

3. Origins of replication Origins of replication Replication Bubbles 2.Replication Bubbles: Hundreds (Eukaryotes) a.Hundreds of replicating bubbles (Eukaryotes). Single(bacteria). b.Single replication fork (bacteria). Bubbles

4. Required components for the DNA replication: 1.dNTPs: dATP, dTTP, dGTP, dCTP (deoxyribonucleoside 5’-triphosphates), (sugar-base + 3 phosphates) 2.DNA template 3.DNA polymerase 4.Mg 2+ (optimizes DNA polymerase activity)

5. Strand Separation Strand Separation: Helicase unwindingseparation 1.Helicase: enzyme which catalyze the unwinding and separation (breaking H-Bonds) of the parental double helix. Single-Strand Binding Proteins 2.Single-Strand Binding Proteins: proteins which attach and help keep the separated strands apart. 3.Topoisomeraserelieves stressDNA molecule 3.Topoisomerase: enzyme which relieves stress on the DNA molecule by allowing free rotation around a single strand. Enzyme DNA Enzyme

6. Priming:Priming: 1.RNA primers primers (RNA) (DNA Polymerase) 1.RNA primers: before new DNA strands can form, there must be small preexisting primers (RNA) present to start the addition of new nucleotides (DNA Polymerase). 2.Primase 2.Primase: enzyme that polymerizes (synthesizes) the RNA Primer. Three main features of the DNA synthesis reaction: DNA polymerase I catalyzes formation of phosphodiester bond between 3’-OH of the deoxyribose (on the last nucleotide) and the 5’-phosphate of the dNTP. Energy for this reaction is derived from the release of two of the three phosphates of the dNTP. DNA polymerase “finds” the correct complementary dNTP at each step in the lengthening process. rate ≤ 800 dNTPs/second low error rate Direction of synthesis is 5’ to 3’

7. DNA elongation:

8. Origin of replication (e.g., the prokaryote example): Begins with double-helix denaturing into single-strands thus exposing the bases. Exposes a replication bubble from which replication proceeds in both directions.

10. Initiation of replication, major elements:  Segments of DNA are called template strands.  Gyrase (a type of topoisomerase) relaxes the supercoiled DNA.  Initiator proteins and DNA helicase binds to the DNA at the replication fork and untwist the DNA using energy derived from ATP (adenosine triphosphate).  (Hydrolysis of ATP causes a shape change in DNA helicase)  DNA primase next binds to helicase producing a complex called a primosome (primase is required for synthesis),  Primase synthesizes a short RNA primer of 10-12 nucleotides, to which DNA polymerase III adds nucleotides.  Polymerase III adds nucleotides 5’ to 3’ on both strands beginning at the RNA primer.  The RNA primer is removed and replaced with DNA by polymerase I, and the gap is sealed with DNA ligase.  Single-stranded DNA-binding (SSB) proteins (>200) stabilize the single-stranded template DNA during the process.

11. Synthesis of the new DNA Strands: Synthesis of the new DNA Strands: 1.DNA PolymeraseRNA primer synthesis of a new DNA strand in the 5’ to 3’ direction 1.DNA Polymerase: with a RNA primer in place, DNA Polymerase (enzyme) catalyze the synthesis of a new DNA strand in the 5’ to 3’ direction. RNAPrimer DNA Polymerase Nucleotide 5’ 3’

12. 1- 1- Leading Strand: synthesized as a single polymer in the 5’ to 3’ direction. RNAPrimer DNA Polymerase Nucleotides 3’5’ 2- 2- Lagging Strand: also synthesized in the 5’ to 3’ direction, but discontinuously. Lagging Strand RNAPrimerDNAPolymerase 3’ 5’ Okazaki Fragment

13. DNA replication is continuous on the leading strand and semidiscontinuous on the lagging strand: Unwinding of any single DNA replication fork proceeds in one direction. The two DNA strands are of opposite polarity, and DNA polymerases only synthesize DNA 5’ to 3’. Solution: DNA is made in opposite directions on each template. Leading strand synthesized 5’ to 3’ in the direction of the replication fork movement. continuous. requires a single RNA primer Lagging strand synthesized 5’ to 3’ in the opposite direction. semidiscontinuous (i.e., not continuous) requires many RNA primers, DNA is synthesized in short fragments.

14. 3 Polymerase III 5’  3’ Leading strand base pairs 5’5’ 5’5’ 3’3’ 3’3’ Helicase + Initiator Proteins ATP SSB Proteins RNA Primer primase 2 Polymerase III Lagging strand Okazaki Fragments 1 RNA primer replaced by polymerase I & gap is sealed by ligase

15. Model of DNA Replication

16. 5.DNA ligase 3’ to 5’ end 5.DNA ligase: a linking enzyme that catalyzes the formation of a covalent bond from the 3’ to 5’ end of joining stands. Example: joining two Okazaki fragments together. Lagging Strand Okazaki Fragment 2 DNA ligase DNA ligase Okazaki Fragment 1 5’ 3’

17. DNA ligase seals the gaps between Okazaki fragments with a phosphodiester bond

18. DNA replication proteins EnzymeFunction in DNA replication DNA Helicase Also known as helix destabilizing enzyme. Unwinds the DNA double helix at the Replication Fork.Replication Fork DNA Polymerase Builds a new duplex DNA strand by adding nucleotides in the 5' to 3' direction. Also performs proof-reading and error correction. There exist many different types of DNA Polymerase, each of which perform different functions in different types of cells. (RNA Polymerase) Primase Provides a starting RNA short primers for DNA polymerase to begin synthesis of the new DNA strand. DNA clamp A protein which prevents elongating DNA polymerases from dissociating from the DNA parent strand. Single-Strand Binding (SSB) Proteins Bind to ssDNA and prevent the DNA double helix from reannealing after DNA helicase unwinds it, thus maintaining the strand separation, and facilitating the synthesis of the nascent strand. TopoisomeraseRelaxes the DNA from its super-coiled nature. DNA Ligase Re-anneals the s and and joins Okazaki Fragments of the lagging strand.Okazaki Fragments

19. Replication of circular DNA in E. coli (3.10): 1.Two replication forks result in a theta-like (  ) structure. 2.As strands separate, positive supercoils form elsewhere in the molecule. 3.Topoisomerases relieve tensions in the supercoils, allowing the DNA to continue to separate.

20. In prokaryotes, there are three main types of DNA polymerase PolymerasePolymerization (5’-3’)Exonuclease (3’-5’) Exonuclease (5’-3’)#Copies IYesYes Yes 400 IIYesYes No ? IIIYesYes No 10-20 3- 5’ exonuclease activity = ability to remove nucleotides from the 3’ end of the chain Important proofreading ability Without proofreading error rate (mutation rate) is 1 x 10 -6 5- 3’ exonuclease activity functions in DNA replication and repair.

21. Eukaryotic enzymes: Five common DNA polymerases from mammals. 1.Polymerase  (alpha): nuclear, DNA replication, no proofreading 2.Polymerase  (beta): nuclear, DNA repair, no proofreading 3.Polymerase  (gamma): mitochondria, DNA repl., proofreading 4.Polymerase  (delta): nuclear, DNA replication, proofreading 5.Polymerase  (epsilon): nuclear, DNA repair (?), proofreading Different polymerases for the nucleus and mtDNA Some polymerases proofread; others do not. Some polymerases used for replication; others for repair. Polymerases vary by species.

23. DNA elongation:

24. Why are RNA primers used in DNA replication DNA Polymerase, the main enzyme involved in DNA synthesis requires a template, and a primer (the -OH group on the THIRD carbon (not the second) of deoxyribose). This -OH group is located on the previous nucleotide. The phosphate group ( -PO4 ) of the incoming nucleotide will be added to the ( -OH ) group of the previous nucleotide. So now for the answer. Since the DNA polymerase needs to "see" a 3 prime hydroxyl group ( -OH ) in order to proceed adding new bases along the template, "How does synthesis start in the first place?" The answer comes from RNA polymerase (the enzyme responsible for synthesizing RNA from a DNA template). At the start of DNA replication, RNA polymerase creates a short temporary primer on the DNA template. Like DNA polymerase, the RNA polymerase requires a template, BUT unlike DNA polymerase, the RNA does NOT need to see that -OH group of the previous nucleotide. After a short RNA primer sequence has been created on the DNA template strand, the RNA polymerase falls off and DNA polymerase can add onto the hydroxyl group (-OH) of the RNA primer and start synthesizing from there. The RNA is removed shortly after the DNA synthesis starts. RNA is used because it is easily made for a temporary purpose such as replicating DNA and then degraded. DNA is used when the cell wants something permanent

25. Replication forks visible in Drosophila

26. Final Step - Assembly into Nucleosomes: As DNA unwinds, nucleosomes must disassemble. Histones and the associated chromatin proteins must be duplicated by new protein synthesis. Newly replicated DNA is assembled into nucleosomes almost immediately. Histone chaperone proteins control the assembly.

27. Concepts and terms to understand: Why are topoisomerase and helicase required? The difference between a template and a primer? The difference between primase and polymerase? What is a replication fork and how many are there? Why are single-stranded binding (SSB) proteins required? How does synthesis differ on leading strand and lagging strand? Which is continuous and discontinuous? What are Okazaki fragments?

28. THANKS