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DNA Replication

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DNA Replication, Replication Fork, Proofreading
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DNA Replication

  1. 1. REPLICATION Fb.com/careeratfoodscience
  2. 2. The Molecular Basis of Inheritance C T A A T CG GC A C G AT AT A T TA C TA 0.34 nm 3.4 nm (a) Key features of DNA structure G 1 nm G (c) Space-filling model T
  3. 3. • Structure was discovered in 1953 by James Watson and Francis Crick
  4. 4. The Structure of DNA • DNA is composed of four nucleotides, each containing: adenine, cytosine, thymine, or guanine. • The amounts of A = T, G = C, and purines = pyrimidines [Chargaff’s Rule]. • DNA is a double-stranded helix with antiparallel strands [Watson and Crick]. • Nucleotides in each strand are linked by 5’-3’ phosphodiester bonds • Bases on opposite strands are linked by hydrogen bonding: A with T, and G with C.
  5. 5. The Basic Principle: Base Pairing to a Template Strand • The relationship between structure and function is manifest in the double helix • Since the two strands of DNA are complementary each strand acts as a template for building a new strand in replication
  6. 6. DNA replication • The parent molecule unwinds, and two new daughter strands are built based on base-pairing rules (a) The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C. (b) The first step in replication is separation of the two DNA strands. (c) Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand. (d) The nucleotides are connected to form the sugar-phosphate backbones of the new strands. Each “daughter” DNA molecule consists of one parental strand and one new strand. A C T A G A C T A G A C T A G A C T A G T G A T C T G A T C A C T A G A C T A G T G A T C T G A T C T G A T C T G A T C
  7. 7. DNA Replication is “Semi-conservative” • Each 2-stranded daughter molecule is only half new • One original strand was used as a template to make the new strand
  8. 8. DNA Replication • The copying of DNA is remarkable in its speed and accuracy • Involves unwinding the double helix and synthesizing two new strands • More than a dozen enzymes and other proteins participate in DNA replication • The replication of a DNA molecule begins at special sites called origins of replication, where the two strands are separated
  9. 9. Origins of Replication • A eukaryotic chromosome may have hundreds or even thousands of replication origins Replication begins at specific sites where the two parental strands separate and form replication bubbles. The bubbles expand laterally, as DNA replication proceeds in both directions. Eventually, the replication bubbles fuse, and synthesis of the daughter strands is complete. 1 2 3 Origin of replication Bubble Parental (template) strand Daughter (new) strand Replication fork Two daughter DNA molecules In eukaryotes, DNA replication begins at many sites along the giant DNA molecule of each chromosome. In this micrograph, three replication bubbles are visible along the DNA of a cultured Chinese hamster cell (TEM). (b)(a) 0.25 µm
  10. 10. Mechanism of DNA Replication • DNA replication is catalyzed by DNA polymerase which needs an RNA primer • RNA primase synthesizes primer on DNA strand • DNA polymerase adds nucleotides to the 3’ end of the growing strand
  11. 11. Mechanism of DNA Replication • Nucleotides are added by complementary base pairing with the template strand • The substrates, deoxyribonucleoside triphosphates, are hydrolyzed as added, releasing energy for DNA synthesis.
  12. 12. The Mechanism of DNA Replication • DNA synthesis on the leading strand is continuous • The lagging strand grows the same general direction as the leading strand (in the same direction as the Replication Fork). However, DNA is made in the 5’- to-3’ direction • Therefore, DNA synthesis on the lagging strand is discontinuous • DNA is added as short fragments (Okasaki fragments) that are subsequently ligated together
  13. 13. DNA polymerase I degrades the RNA primer and replaces it with DNA
  14. 14. The Mechanism of DNA Replication • Many proteins assist in DNA replication • DNA helicases unwind the double helix, the template strands are stabilized by other proteins • Single-stranded DNA binding proteins make the template available • RNA primase catalyzes the synthesis of short RNA primers, to which nucleotides are added. • DNA polymerase III extends the strand in the 5’-to-3’ direction • DNA polymerase I degrades the RNA primer and replaces it with DNA • DNA ligase joins the DNA fragments into a continuous daughter strand
  15. 15. Enzymes in DNA replication Helicase unwinds parental double helix Binding proteins stabilize separate strands DNA polymerase III binds nucleotides to form new strands Ligase joins Okazaki fragments and seals other nicks in sugar- phosphate backbone Primase adds short primer to template strand DNA polymerase I (Exonuclease) removes RNA primer and inserts the correct bases
  16. 16. Binding proteins prevent single strands from rewinding. Helicase protein binds to DNA sequences called origins and unwinds DNA strands. 5’ 3’ 5’ 3’ Primase protein makes a short segment of RNA complementary to the DNA, a primer. 3’5’ 5’3’ Replication
  17. 17. Overall direction of replication 5’3’ 5’ 3’ 5’ 3’ 3’5’ DNA polymerase enzyme adds DNA nucleotides to the RNA primer. Replication
  18. 18. DNA polymerase enzyme adds DNA nucleotides to the RNA primer. 5’ 5’ Overall direction of replication 5’ 3’ 5’ 3’ 3’ 3’ DNA polymerase proofreads bases added and replaces incorrect nucleotides. Replication
  19. 19. 5’ 5’3’ 5’ 3’ 3’ 5’ 3’ Overall direction of replication Leading strand synthesis continues in a 5’ to 3’ direction. Replication
  20. 20. 3’5’ 5’ 5’3’ 5’ 3’ 3’ 5’ 3’ Overall direction of replication Okazaki fragment Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments. Replication
  21. 21. 5’ 5’ 5’3’ 5’ 3’ 3’ 5’ 3’ Overall direction of replication 3’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments. Okazaki fragment Replication
  22. 22. 5’ 5’ 3’ 5’ 3’ 3’ 5’ 3’ 3’ 5’ 5’3’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments. Replication
  23. 23. 3’ 5’ 3’ 5’ 5’ 3’ 5’ 3’ 3’ 5’ 5’3’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments. Replication
  24. 24. 5’ 5’ 3’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ Exonuclease activity of DNA polymerase I removes RNA primers. Replication
  25. 25. Polymerase activity of DNA polymerase I fills the gaps. Ligase forms bonds between sugar-phosphate backbone. 3’ 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ Replication
  26. 26. Replication Fork Overview
  27. 27. Proofreading • DNA must be faithfully replicated…but mistakes occur – DNA polymerase (DNA pol) inserts the wrong nucleotide base in 1/10,000 bases • DNA pol has a proofreading capability and can correct errors – Mismatch repair: ‘wrong’ inserted base can be removed – Excision repair: DNA may be damaged by chemicals, radiation, etc. Mechanism to cut out and replace with correct bases
  28. 28. Mutations • A mismatching of base pairs, can occur at a rate of 1 per 10,000 bases. • DNA polymerase proofreads and repairs accidental mismatched pairs. • Chances of a mutation occurring at any one gene is over 1 in 100,000 • Because the human genome is so large, even at this rate, mutations add up. Each of us probably inherited 3-4 mutations!
  29. 29. Proofreading and Repairing DNA • DNA polymerases proofread newly made DNA, replacing any incorrect nucleotides • In mismatch repair of DNA, repair enzymes correct errors in base pairing • In nucleotide excision DNA repair nucleases cut out and replace damaged stretches of DNA Nuclease DNA polymerase DNA ligase A thymine dimer distorts the DNA molecule. 1 A nuclease enzyme cuts the damaged DNA strand at two points and the damaged section is removed. 2 Repair synthesis by a DNA polymerase fills in the missing nucleotides. 3 DNA ligase seals the Free end of the new DNA To the old DNA, making the strand complete. 4
  30. 30. •Thank you

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