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UNIT 2

UNIT 2. DNA Replication. Objectives Discuss experimental evidence supporting semi- conservative mechanism of DNA replication Explain DNA replication in Prokaryotes and Eukaryotes Define mutation Identify different types of mutations and their effects on the protein products produced.

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UNIT 2

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  1. UNIT 2 DNA Replication

  2. Objectives Discuss experimental evidence supporting semi- conservative mechanism of DNA replication Explain DNA replication in Prokaryotes and Eukaryotes Define mutation Identify different types of mutations and their effects on the protein products produced

  3. Eukaryotic genes have interrupted coding sequences. • That is, there are long sequences of bases within the protein-coding sequences of the gene that do not code for amino acids in the final protein product. • The nocoding regions within the gene are called introns (intervening sequences). • The exons (expressed sequences) which are part of the protein-coding sequence.

  4. A typical eukaryotic gene may have multiple exons and introns and the numbers are quite variable. Eg. the β-globulin gene has 2 and the ovalbumin gene of egg white has 7. • In many cases the lengths of the introns are much greater than those of the exon sequences. • For instance the ovalbumin gene contains about 7700 base pairs, 1859 of them in exons.

  5. DNA Replication Three theories were suggested: • Conservative replication • intact the original DNA molecule and generate a completely new molecule. • Dispersive replication • produce two DNA molecules with sections of both old and new DNA interspersed along each strand. • Semi-conservative replication • produce molecules with both old and new DNA - each molecule would be composed of one old strand and one new one.

  6. DNA Replication is semi-conservativeExperimental Proof • (1957) Mathew Meselson and Franklin Stahl grew the bacterium Escherichia coli on medium that contained 15N in the form of ammonium chloride. • The 15N became incorporated into DNA (nitrogenous bases). • The resulting heavy nitrogen-containing DNA molecules were extracted from some of the cells.

  7. DNA Replication is semi-conservativeExperimental Proof • When subject to density gradient centrifugation, they accumulated in the high-density region of the gradient. • The rest of the bacteria were transferred to a new growth medium in which ammonium chloride contained the naturally abundant, lighter 14N isotope.

  8. DNA Replication is semi-conservativeExperimental Proof • The newly synthesized strands were expected to be less dense since they incorporated bases containing the lighter 14N isotope. • The DNA from cells isolated after one generation had an intermediate density, indicating that they contained half as many 15N isotope as the parent DNA. • This finding supported the semi-conservative model - each double helix would contain one previously synthesized strand and a newly synthesized strand.

  9. DNA Replication is semi-conservativeExperimental Proof • It is also consistent with the dispersive model which would yield one class of molecules, all with intermediate density. • It was inconsistent with the conservative model which predicted that there would be two classes of double-stranded molecules, those with two heavy strands and those with two light strands. • After another cycle of cell division in the medium with the lighter 14N isotope, two types of DNA appeared in the density gradient.

  10. DNA Replication is semi-conservativeExperimental Proof • One with hybrid DNA helices ( one strand 15N isotope and the other strand 14N), whereas the other contained only strands of the light isotope. • This finding refuted the dispersive model, which predicted that all stands should have intermediate density. • It however supported the semiconservative method which predicted that each parent strand would act as a template for the synthesis of new strands.

  11. Animation of DNA Replication Experimental Proof • http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter14/animations.html Tutorial • http://www.sumanasinc.com/webcontent/animations/content/meselson.html

  12. DNA Replication in Bacteria • In general, DNA is replicated by: • uncoiling of the helix • strand separation by breaking of the hydrogen bonds between the complementary strands • synthesis of two new strands by complementary base pairing Replication begins at a specific site in the DNA called the origin of replication (ori)

  13. DNA Replication in Bacteria DNA replication is bidirectional from the origin of replication DNA replication occurs in both directions from the origin of replication in the circular DNA found in most bacteria.

  14. DNA Replication in Bacteria • To begin DNA replication, unwinding enzymes called DNA helicases cause the two parent DNA strands to unwind and separate from one another at the origin of replication to form two "Y"-shaped replication forks. • These replication forks are the actual site of DNA copying

  15. Replication Fork

  16. Animation of Replication Fork • http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter14/animations.html#

  17. DNA Replication in Bacteria • Helix destabilizing proteins bind to the single-stranded regions so the two strands do not rejoin • Enzymes called topoisimerases produce breaks in the DNA and then rejoin them in order to relieve the stress in the helical molecule during replication.

  18. DNA Replication in Bacteria • As the strands continue to unwind in both directions around the entire DNA molecule, new complementary strands are produced by the hydrogen bonding of free DNA nucleotides with those on each parent strand • As the new nucleotides line up opposite each parent strand by hydrogen bonding, enzymes called DNA polymerases join the nucleotides by way of phosphodiester bonds.

  19. DNA Replication in Bacteria • The nucleotides lining up by complementary base pairing are deoxynucleoside triphosphates • As the phosphodiester bond forms between the 5' phosphate group of the new nucleotide and the 3' OH of the last nucleotide in the DNA strand, two of the phosphates are removed providing energy for bonding

  20. DNA Replication by Complementary Base Pairing

  21. Animation of How Nucleotides are added • http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter14/animations.html#

  22. DNA replication in a 5' to 3' direction

  23. DNA Replication in Bacteria • DNA replication is more complicated than this because of the nature of the DNA polymerases. • DNA polymerase enzymes are only able to join the phosphate group at the 5' carbon of a new nucleotide to the hydroxyl (OH) group of the 3' carbon of a nucleotide already in the chain. • As a result, DNA can only be synthesized in a 5' to 3' direction while copying a parent strand running in a 3' to 5' direction.

  24. DNA Replication in Bacteria • The two strands are antiparallel – • one parent strand - the one running 3' to 5' is called the leading strand can be copied directly down its entire length • the other parent strand - the one running 5' to 3' is called the lagging strand must be copied discontinuously in short fragments – Okazaki fragments of around 100-1000 nucleotides each as the DNA unwinds.

  25. DNA Replication in Bacteria • DNA polymerase enzymes cannot begin a new DNA chain from scratch. • can only attach new nucleotides onto 3' OH group of a nucleotide in a preexisting strand. • To start the synthesis of the leading strand and each DNA fragment of the lagging strand, an RNA polymerase complex called a primosome or primase is required. • The primase is capable of joining RNA nucleotides without requiring a preexisting strand of nucleic acid - forms what is called an RNA primer

  26. RNA primer

  27. DNA Replication in Bacteria • After a few nucleotides are added, primase is replaced by DNA polymerase. • DNA polymerase can now add nucleotides to the 3'end of the short RNA primer. • The primer is later degraded and filled in with DNA.

  28. DNA Replication in Bacteria Bacteria have 5 known DNA polymerases: • Pol I: • DNA repair • has 5'→3' (Polymerase) activity • both 3' → 5' (proof reading) and 5' → 3' exonuclease activity (in removing RNA primers).

  29. DNA polymerase I is not the replicative polymerase: 1. The enzyme is too slow!adds dNTPs at a rate of 20 nt/sec. So it would require 460,000 sec (= 7667 min = 128 hr = 5.3 days) to replicate the E. coli chromosome! Too slow for an organism which can divide every 20 mins. 2. The enzyme is too abundantThere are 400 molecules per E. coli cell. This is excessive given that there are generally only 2 replication forks per cell. 3. The enzyme is not processive enoughDNA polymerase I dissociates after catalysing the incorporation of 20-50 nucleotides.

  30. DNA Replication in Bacteria • Pol II: • involved in repair of damaged DNA • has 3' → 5' exonuclease activity. Proof that this is not the main polymerase: 1. Strains lacking the gene show no defect in growth or replication. 2. Synthesis of Pol II is induced during the stationary phase of cell growth - a phase in which little growth and DNA synthesis occurs. But DNA can accumulate damage such as short gaps 3. Pol II has a low error rate but it is much too slow to be of any use in normal DNA synthesis.

  31. DNA Replication in Bacteria • Pol III: • the main polymerase in bacteria (elongates in DNA replication) • has 3' → 5' exonuclease proofreading ability. • is the principal replicative enzyme Proof of function: 1. is highly processive 2. catalyses polymerization at a high rate.

  32. There are two forms of the enzyme. Core enzyme - consists of only those subunits that are required for the basic underlying enzymatic activity: alpha (a), epsilon (e) and theta (q). Holoenzyme- the fully functional form of an enzyme, complete with all of its necessary accessory subunits. The DNA polymerase III holoenzyme consists of the core enzyme, the b sliding clamp and the clamp-loading complex.

  33. DNA Replication in Bacteria • Pol IV and Pol V: • Are Y-family DNA polymerases • participates in bypassing DNA damage

  34. DNA Replication in Bacteria

  35. Animation of bidirectional replication of DNA • http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter11/animations.html#

  36. DNA Replication in Eukaryotes • multiple origins of replication in eukaryotes • human genome about 30,000 origins • each origin produces two replication forks • moving in opposite direction

  37. DNA Replication in Eukaryotes

  38. DNA Replication in Eukaryotes Eukaryotes have at least 15 DNA Polymerases: • Pol α : act as a primase (synthesizing an RNA primer), elongates the primer • Pol β : repairs DNA, (excision repair and gap-filling). • Pol γ: Replicates and repairs mitochondrial DNA and has proofreading 3' → 5' exonuclease activity. • Pol δ: Highly processive and has proofreading 3' → 5' exonuclease activity, reposible for replication of lagging strand. • Pol ε: Highly processive and has proofreading 3' → 5' exonuclease activity, reponsible for replication of leading strand. • η, ι, κ, Rev1 and Pol ζ are involved in the bypass of DNA damage. • θ, λ, φ, σ, and μ are not as well characterized: • There are also others, but the nomenclature has become quite jumbled.

  39. DNA Replication in Eukaryotes • the polymerases that deal with the elongation are Pol α,Pol ε,Polδ. • Pol α : forms a complex to act as a primase (synthesizing an RNA primer), and then elongates that primer with DNA nucleotides. • After around 20 nucleotides elongation by Pol α is taken over by Pol ε (on the leading strand) and δ (on the lagging strand). • Other enzymes are responsible for primer remover in Eukaryotes as none of their polymerases have 5′→3′ exonuclease activity

  40. DNA Replication in Eukaryotes

  41. DNA damage bypass • All organisms need to deal with the problems that arise when a moving replication fork encounters damage in the template strand. • The best way to deal with this situation is to repair the damage by an excision mechanisms. • In some cases, however, the damage may not be repairable, or the advancing replication fork may already have unwound the parental strands, thus preventing excision mechanisms from using the complementary strand as template for repair, or excision repair may not yet have had an opportunity to repair the damage.

  42. DNA damage bypass • It is important for the cell to be able to move replication forks past unrepaired damage: • Long-term blockage of replication forks leads to cell death. • Replication of damaged DNA provides a sister chromatid that can be used as template for subsequent repair by homologous recombination. • Replication fork bypass mechanisms cannot, strictly speaking, be considered examples of DNA repair, because the damage is left in the DNA, at least temporarily.

  43. Rate of Replication • In prokaryotes replication proceeds at about 1000 nucleotides per second, and thus is done in no more than 40 minutes. • In Eukaryotes replication takes proceeds at 50 nucleotides per second, and is completed in 60 minutes.

  44. Mutations • changes in the nucleotide sequence of the DNA. • organisms have special systems of enzymes that can repair certain kinds of alterations in the DNA. • once the DNA sequence has been changed, DNA replication copies the altered sequence just as it would copy a normal sequence. • provide the variation necessary for evolution to happen in a given species.

  45. Types of Mutations Somatic mutations • Occurs in cells not dedicated to sexual reproduction • The mutant genes disappear when the cell in which it occurred dies and can only be passed on through asexual reproduction. Germline mutations • found in every cell descended from the zygote to which that mutant gamete contributed. • If an adult is successfully produced, every one of its cells will contain the mutation.

  46. Types of Mutations Single-base Substitution/point mutation • exchanges one base for another. • If one purine [A or G] or pyrimidine [C or T] is replaced by the other, the substitution is called atransition. • If a purine is replaced by a pyrimidine or vice-versa, the substitution is called atransversion. Original: The fat cat ate the wee rat Point Mutation: The fat hat ate the wee rat

  47. Types of Mutations Point mutations continued • A change in a codon to one that encodes a different amino acid and cause a small change in the protein produced = missense mutation. • Example sickle-cell disease A → T at the 17th nt of the gene for the beta chain of hemoglobin changes the codon GAG (glutamic acid) to GTG (valine) Therefore: 6th amino acid glutamic acid → valine

  48. Missense mutation

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