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This is a power-point presentation on DNA structure and Dna replication

This is a power-point presentation on DNA structure and Dna replication

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  • 1. GUMEDE ESTHER NTOMBIFUTHI 3RD YEAR UNIVERSITY OF JOHANNESBURG 2014
  • 2. NUCLEIC ACIDS :
  • 3. DNA Structure DNA consists of two molecules that are arranged into a ladder-like structure called a Double Helix. A molecule of DNA is made up of millions of tiny subunits called Nucleotides. Each nucleotide consists of: 1. Phosphate group 2. Pentose sugar-Deoxyribose 3. Nitrogenous base
  • 4. Nucleotides Phosphate Nitrogenous Base Pentose Sugar
  • 5. DNA Structure Helps Explain How It Duplicates DNA is made up of two nucleotide strands held together by hydrogen bonds Hydrogen bonds between two strands are easily broken Each single strand then serves as template for new strand
  • 6. DEOXYRIBONUCLEIC ACID DNA usually exists as a double-stranded structure, with both strands coiled together to form the characteristic double-helix. Each single strand of DNA is a chain of four types of nucleotides having the bases: Adenine Cytosine Guanine Thymine 03/07/14 Pranabjyoti Das 6
  • 7. Nucleotides The phosphate and sugar form the backbone of the DNA molecule, whereas the bases form the “rungs”. There are four types of nitrogenous bases.
  • 8. Orientation of DNA on the sugar ring are The carbon atoms numbered for reference. The 5’ and 3’ hydroxyl groups (highlighted on the left) are used to attach phosphate groups.  The directionality of a DNA strand is due to the orientation of the phosphate-sugar backbone.
  • 9. Nucleotides A Adenine C Cytosine T Thymine G Guanine
  • 10. Nucleotides Each base will only bond with one other specific base. Adenine (A) Thymine (T) Cytosine (C) Guanine (G) Form a base pair. Form a base pair.
  • 11. DNA Structure A gene is a section of DNA that codes for a protein. Each unique gene has a unique sequence of bases. This unique sequence of bases will code for the production of a unique protein. It is these proteins and combination of proteins that give us a unique phenotype.
  • 12. DNA Gene Protein Trait
  • 13. A Nucleoside is a combination of Pentose sugar & Nitrogen Base A Nucleotide is a combination of nucleoside & Phosphoric Acid 03/07/14 Pranabjyoti Das 13
  • 14. P T A 5’ C G P 3’ P DNA has directionality. P P P C Two nucleotide chains together wind into a helix. G A P P T P P G Hydrogen bonds between paired bases hold the two DNA strands together. C P P G 3’ C DNA strands are antiparallel. P P A sugar and phosphate “backbone” connects nucleotides in a chain. 5’
  • 15. DNA BACKBONE 03/07/14 Pranabjyoti Das 15
  • 16. Nucleotides are matched between strands through hydrogen bonds to form base pairs. Adenine pairs with thymine and cytosine pairs with guanine 03/07/14 Pranabjyoti Das 16
  • 17. These terms refer to the carbon atom in deoxyribose to which the next phosphate in the chain attaches. Directionality has consequences in DNA synthesis, because DNA polymerase can synthesize DNA in only one direction by adding nucleotides to the 3' end of a DNA strand. 03/07/14 Pranabjyoti Das 17
  • 18. The pairing of bases in DNA through hydrogen bonding means that the information contained within each strand is redundant. The nucleotides on a single strand can be used to reconstruct nucleotides on a newly synthesized partner strand. 03/07/14 Pranabjyoti Das 18
  • 19. Functions DNA is used to store genetic information It is replicated before cell division DNA is very important so it is stored in the nucleus. It never leaves the nucleus Your DNA stores the code for your proteins, which exhibit your “traits” The DNA gets converted to RNA in order to move out into the cytoplasm
  • 20. 03/07/14 Pranabjyoti Das 20
  • 21. DNA replication is a biological process that occurs in all living organisms and copies their exact DNA. It is the basis for biological inheritance. 03/07/14 Pranabjyoti Das 21
  • 22.  Each old strand stays intact  Each new DNA molecule is half “old” and half “new” Fig. 1-7, p.212
  • 23. The first major step for the DNA Replication to take place is the breaking of hydrogen bonds between bases of the two antiparallel strands. The unwounding of the two strands is the starting point. The splitting happens in places of the chains which are rich in A-T. That is because there are only two bonds between Adenine and Thymine. There are three hydrogen bonds between Cytosine and Guanine.  03/07/14 Pranabjyoti Das 23
  • 24. Helicase is the enzyme that splits the two strands. The structure that is created is known as "Replication Fork". 03/07/14 Pranabjyoti Das 25
  • 25. 03/07/14 In order for DNA replication to begin, the double stranded DNA helix must first be opened. The sites where this process first occurs are called replication origins. Helicase unwinds the two single strands 26 Pranabjyoti Das
  • 26. Replication 3’ 3’ 5’ 5’ 3’ 5’ 3’ 5’ Helicase protein binds to DNA sequences called origins and unwinds DNA strands. Binding proteins prevent single strands from rewinding. Primase protein makes a short segment of RNA complementary to the DNA, a primer.
  • 27. Overall direction of replication 3’ 3’ 5’ 5’ 3’ 5’ 3’ 5’ DNA polymerase enzyme adds DNA nucleotides to the RNA primer.
  • 28. Overall direction of replication 3’ 3’ 5’ 5’ 3’ 5’ Leading strand synthesis continues in a 5’ to 3’ direction. 3’ 5’
  • 29. Overall direction of replication 3’ 3’ 5’ 5’ Okazaki fragment 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. 3’ 5’
  • 30. rk o F o n The replication fork is a structure that ti forms within the nucleus during DNA ca i pl replication. It is created by helicases, e R which break the hydrogen bonds holding the two DNA strands together. The resulting structure has two branching "prongs", each one made up of a single strand of DNA. These two strands serve as the template for the leading and lagging strands, which will be created as DNA polymerase matches complementary nucleotides to the templates; The templates may be properly referred to as the leading strand template and the lagging strand template 03/07/14 Pranabjyoti Das 31
  • 31. 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ DNA polymerase enzyme adds DNA nucleotides to the RNA primer. DNA polymerase proofreads bases added and replaces incorrect nucleotides.
  • 32. 3’ 5’ 3’ 5’ 3’ 5’ 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.
  • 33. 3’ 3’ 5’ 5’ Okazaki fragment 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. 3’ 5’
  • 34. 3’ 5’ 3’ 5’ 3’ 5’ 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.
  • 35. 5’ 3’ 3’ 5’ 3’ 3’5’ 5’ 3’5’ 3’ 5’ Exonuclease enzymes remove RNA primers.
  • 36. 3’ 3’ 5’ 3’ 5’ 3’5’ 3’ 5’ Exonuclease enzymes remove RNA primers. Ligase forms bonds between sugar-phosphate backbone.
  • 37. One of the most important steps of DNA Replication is the binding of RNA Primase in the initiation point of the 3'-5' parent chain.  RNA Primase can attract RNA nucleotides which bind to the DNA nucleotides of the 3'-5' strand due to the hydrogen bonds between the bases. RNA nucleotides are the primers (starters) for the binding of DNA nucleotides.  03/07/14 Pranabjyoti Das 38
  • 38. In the lagging strand the DNA Pol IExonuclease- reads the fragments and removes the RNA Primers. The gaps are closed with the action of DNA Polymerase which adds complementary nucleotides to the gaps and DNA Ligase which acts as a glue to attach the phosphate to the sugar by forming phosphodiester bond. 03/07/14 Pranabjyoti Das 39
  • 39. Enzymes in Replication Enzymes (Helicases) unwind the two strands DNA polymerase needed for the synthesis of complementary strand DNA ligase joins pieces of the lagging strand together
  • 40. Helicase unwinds parental double helix DNA polymerase binds nucleotides to form new strands Binding proteins stabilize separate strands Exonuclease removes RNA primer and inserts the correct bases Primase adds short primer to template strand Ligase joins Okazaki fragments and seals other nicks in sugarphosphate backbone
  • 41. DNA Replication models There are three possible models that describe the accurate creation of the daughter chains: Semiconservative Replication Conservative Replication Dispersive Replication
  • 42. Figure 11.2 11-6
  • 43. Replicatoin can’t just start: 1. All DNA polymerases need a primer 2. The primer can be a piece of RNA or DNA 3. It must be “base-paired” with the “template” and with primer 3’OH Thus: 5’ 3’ 3’ Synthesis direction 5’ Template strand
  • 44. “Proof-reading” is essential at DNA replication •What is proof-reading? 1. If there is a wrong base built in, then there is no base paring possible. 2. The DNA polymerase can’t continue on building in the next base. 3. The DNA polymerase removes the “wrong” base and starts over
  • 45. Klenow Fragment (of pol I) (The proof-reading activity)
  • 46.  Bacterial DNA polymerases may vary in their subunit composition  However, they have the same type of catalytic subunit Structure resembles a human right hand Template DNA thread through the palm; Thumb and fingers wrapped around the DNA
  • 47. Proteins involved in E. coli replication Terms to be known
  • 48. DNA replication mistakes The most errors in DNA sequence occur during replication. Reparation takes place after replication is finished DNA polymerases can get the right sequence from the complementary strand and repair, along with DNA ligase, the wrong bases.
  • 49. Is a method in which multiple repetitions of DNA replication are performed in a test tube. Mix in test tube: DNA template DNA to be amplified Primers one complementary to each strand Nucleotides dATP,d GTP, dCTP, and dTTP DNA polymerase heat stable form from thermophilic bacteria
  • 50. DNA template is denatured with heat to separate strands. 5’ 3’ G C 5’ A T A T C G T A A T G C C G G C 3’
  • 51. DNA template is denatured with heat to separate strands. 5’ 3’ G A C T A G C G C 5’ A T T G A T C G C 3’
  • 52. Each DNA primer anneals, binding to its complementary sequence on the template DNA 5’ 3’ G C A T A T 5’ C T A G C G 3’ 5’ 3’ C 5’ T T G A T G C C G G C 3’
  • 53. DNA polymerase creates a new strand of DNA complementary to the template DNA starting from the primer. 3’ G C A T A T C G T A A T G C C G G C 3’ 5’ 5’ 3’ G C 5’ 5’ A T A T C G T A A T G C C G G C 3’
  • 54. Denaturation DNA template is denatured with high heat to separate strands. Annealing Each DNA primer anneals, binding to its complementary sequence on the template DNA Extension DNA polymerase creates a new strand of DNA complementary to the template DNA starting from the primer. Multiple rounds of denaturation-annealing-extension are performed to create many copies of the template DNA between the two primer sequences.
  • 55. Visualization of PCR products
  • 56. agarose gel-electrophoresis of DNA Top Negative pole DNA is Negatively charged Small molecules dissolve faster separation based on size Bottom Positive pole Slab of agarose gel (ethidium bromide staining)