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DNA Replication Senior Biology Mrs. Brunone

DNA Replication Senior Biology Mrs. Brunone. DNA – Structure

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DNA Replication Senior Biology Mrs. Brunone

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  1. DNA Replication Senior Biology Mrs. Brunone

  2. DNA – Structure • A simple yet elegant structure – a double helix with a sugar phosphate “backbone” linked to 4 types of nucleotides on the inside that are paired according to basic rules. Amazingly this simple molecule has the capacity to specify Earth’s incredible biological diversity. • The double-stranded structure suggests a mode of copying (replication) and the long “strings” of the 4 bases encode biological life. • The human genome is just 3.5 billion base pairs and greater than 95% is considered to be non-coding (or “junk”).

  3. History Of DNA Research • Summary • DNA replication is semi-conservative (Meselson-Stahl, 1958). • Replication requires a DNA polymerase, a template, a primer and the 4 nucleotides and proceeds in a 5’ to 3’ direction (Kornberg, 1957). • Replication is semi-discontinuous (continuous on leading strand and discontinuous on lagging strand) and requires RNA primers (Okazaki’s, 1968). • Lagging strand synthesis involves Okazakifragments.

  4. Replication as a Process 1. Double-stranded DNA unwinds. 2. The junction of the unwound molecules is a replication fork. 3. A new strand is formed by pairing complementary bases with the old strand. 4. Two molecules are made. Each has one new and one old DNA strand. “Semi-conservative”

  5. DNA Replication is Semi-discontinuous Continuous synthesis Discontinuous synthesis

  6. DNA SYNTHEIS REACTION 5' end of strand P P Base Base CH2 CH2 O O P P CH2 CH2 Base Base products O O H20 + 3' P P P P OH P Synthesis reaction Base CH2 P O CH2 5' Base O OH 3' 3' end of strand OH

  7. How is DNA primed? Primase: • Makes initial nucleotide (RNA primer) to which DNA polymerase III attaches • New strand initiated by adding nucleotides to RNA primer • RNA primer later replaced with DNA

  8. Proteins Involved in DNA Replication in E. coli

  9. Primase adds short primer to template strand Binding proteins stabilise separate strands Helicase unwinds parental double helix Ligase joins Okazaki fragments and seals other nicks in sugar-phosphate backbone DNA polymerase I (Exonuclease) removes RNA primer and inserts the correct bases DNA polymerase binds nucleotides to form new strands Enzymes in DNA replication

  10. 3’ 5’ 3’ 5’ 5’ 3’ 3’ 5’ Binding proteins prevent single strands from rewinding. Primase protein makes a short segment of RNA complementary to the DNA, a primer. Replication Helicase protein binds to DNA sequences called origins and unwinds DNA strands.

  11. Overall direction of replication 3’ 3’ 5’ 5’ 3’ 5’ 3’ 5’ Replication DNA polymerase enzyme adds DNA nucleotides to the RNA primer.

  12. Overall direction of replication 3’ 5’ 3’ 5’ 3’ 3’ 5’ 5’ Replication DNA polymerase enzyme adds DNA nucleotides to the RNA primer. DNA polymerase proofreads bases added and replaces incorrect nucleotides.

  13. Overall direction of replication 3’ 3’ 5’ 5’ 3’ 5’ 3’ 5’ Replication Leading strand synthesis continues in a 5’ to 3’ direction.

  14. Overall direction of replication 3’ 3’ 5’ 5’ Okazaki fragment 3’ 5’ 3’ 5’ 5’ 3’ Replication Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.

  15. Replication Overall direction of replication 3’ 3’ 5’ 5’ Okazaki fragment 3’ 5’ 3’ 5’ 3’ 5’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.

  16. Replication 3’ 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ 3’ 5’ 5’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.

  17. 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ Replication 3’ 3’ 5’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.

  18. 3’ 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ 3’ 5’ 5’ Replication Exonuclease activity of DNA polymerase I removes RNA primers.

  19. 3’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ 5’ Replication Polymerase activity of DNA polymerase I fills the gaps. Ligase forms bonds between sugar-phosphate backbone.

  20. DNA REPLICATION 3 Pol III synthesises leading strand 2 1 Helicase opens helix Topoisomerase nicks DNA to relieve tension from unwinding Primase synthesises RNA primer 4 5 Pol I excises RNA primer; fills gap 6 7 Pol III elongates primer; produces Okazaki fragment DNA ligase links Okazaki fragments to form continuous strand

  21. DNA Synthesis •Synthesis on leading and lagging strands •Proofreading and error correction during DNA replication •Simultaneous replication occurs via looping of lagging strand

  22. Simultaneous Replication Occurs via Looping of the Lagging Strand •Helicase unwinds helix •SSBPs prevent closure •DNA gyrase reduces tension •Association of core polymerase with template •DNA synthesis •Not shown: pol I, ligase

  23. BIDIRECTIONALREPLICATION Origin 3’ 5’ 5’ 3’ ori ter Replication Termination of the Bacterial Chromosome

  24. ori ter Replication Forks Procaryotic (Bacterial) Chromosome Replication Bidirectional Replication Produces a Theta Intermediate

  25. Summary • DNA replication proteins: • DNA Pol III • DNA Pol I • DNA Ligase • Primase • Helicase • SSB • Gyrase • Exonuclease (DNAP II)

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