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Fundamentals of Nucleic Acid Biochemistry: DNA

Fundamentals of Nucleic Acid Biochemistry: DNA. Donna C. Sullivan, PhD Division of Infectious Diseases University of Mississippi Medical Center. Defines the relationships between DNA, RNA, and protein in the transmission of genetic information into functional units of biological activity. DNA.

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Fundamentals of Nucleic Acid Biochemistry: DNA

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  1. Fundamentals of Nucleic Acid Biochemistry: DNA Donna C. Sullivan, PhD Division of Infectious Diseases University of Mississippi Medical Center

  2. Defines the relationships between DNA, RNA, and protein in the transmission of genetic information into functional units of biological activity. DNA RNA Protein The Central Dogma ofMolecular Biology

  3. THE GENOME

  4. STRUCTURE OF NUCLEIC ACIDS • Primary structure • Polymers of nucleotides • Nucleotides • Phosphate group • Sugar • Base

  5. TWO TYPES OF SUGAR IN NUCLEIC ACIDS Both retain a 3’ hydroxyl group

  6. single-ringed double-ringed Purines & Pyrimidines Diagrams Source: http://www.mun.ca/biology/scarr/2250_DNA_biochemistry.htm

  7. Structure of Nucleotides

  8. DNA AND RNA: BASIC STRUCTURE

  9. Repeating Nucleotide Subunits In DNA and RNA

  10. STRUCTURE OF DNA • SUGAR • Deoxyribose • Phosphate group • Nitrogen containing base • Adenine • Guanine • Cytosine • Thymidine

  11. Polymerized Nucleotides

  12. Directionality: 5’ to 3’

  13. DOUBLE HELIX OF DNA

  14. CONFORMATIONS OF DNA

  15. THE DOUBLE HELIX: FORMS • B form (Major form) • Right handed • Two helical grooves which allow protein binding • A form • Found in RNA-DNA or RNA-RNA helices • More compact than B form • Z form (Zig-Zag) • Left handed • Composed of alternating G and C residues

  16. GROOVES OF DNA

  17. DNA MOLECULAR CONFORMATIONS • Linear ds DNA found in eukaryotic cell • Other conformations found in • Prokaryotic cells: Circular ds DNA • Viruses: • Circular ds DNA (Adenoviruses, SV40) • Linear ss DNA (Parvoviruses) • Circular partially ds DNA (HBV)

  18. DNA SYNTHESIS • Semiconservative • “New” DNA contains one “old” strand plus one “new” strand • Bidirectional • Each of the “old” strands is copied • Each strand is copied simultaneously • Initiates at a common site: origin of replication

  19. DNA REPLICATION

  20. DNA REPLICATION:Semi-conservative

  21. DNA REPLICATION ORIGIN • Replication of DNA begins at specific sites • E. coli ori (1): ~240 bp • Yeast ori: 400 on 17 chromosomes • Contain multiple short repeated sequences • Repeat units recognized by multimeric origin binding proteins • Usually contain an AT rich region

  22. DNA POLYMERASES • Prokaryotic DNA Polymerases • DNA pol I • DNA pol II • DNA pol III • Eukaryotic DNA Polymerases • DNA pol  • DNA pol  • DNA pol  (Two additional DNA pols  and )

  23. CHARACTERISTICS OF DNA POLYMERASES • Some DNA Polymerases have proof-reading function • Can not separate (uncoil or unwind) DNA • Must have a primer to initiate replication, primers synthesized by RNA Polymerases • Catalyze nucleotide addition at the 3’ OH, strands only grow in 5’ to 3’ direction

  24. DNA REPLICATION:5’ 3’ DIRECTION OF SYNTHESIS

  25. OTHER ENZYMES AT THE INTITIATION SITE • DNA gyrase: unwinds supercoils • DNA helicase: separates double helix • Single stranded DNA binding protein (ss DBP) • Primase: a type of RNA Polymerase • Topoisomerase

  26. DNA REPLICATION: REPLICATION FORK

  27. UNWINDING SUPERCOILS • Supercoiling occurs when: • Two ends of DNA are fixed (ie, circular) • DNA is in long helical structure (ie, large chromosome) • Supercoiling occur during: • Replication of DNA • Transcription of RNA from DNA • Binding by some types of protein • Supercoiling is relieved by helicases, topoisomerases

  28. “LEADING” AND “LAGGING” STRANDS OF DNA • “Leading” strand of daughter DNA • Synthesis occurs continuously from a single RNA primer at its 5’ end • “Lagging” strand of daughter DNA • Synthesis occurs discontinuously from multiple RNA primers that are formed on parental strand as each new region of DNA is exposed at the growing fork • Okazaki fragments • Joined by DNA ligase to form continuous DNA

  29. Termination • Once the new strands are complete, the molecules rewind automatically in order to regain their stable helical structure.

  30. Termination • A problem is created once the RNA primer is removed from the 5’ end of each daughter strand, there is no adjacent fragment for which new nucleotides can be added to fill this gap, resulting in a slightly shorter daughter chromosomes. • This occurrence is not a problem in circular DNA, but human cells loose about 100 base pairs from each end of each chromosome with each replication

  31. Termination • This loss of genetic material could result in critical code being eliminated, however there are buffer zones of repetitive nucleotide sequences, called the telomeres. • In humans the sequence is TTAGGG repeated several thousand times.

  32. Telomeres and Cell Death • Their erosion does not affect cell function, but protects against lost of important genetic material. • The erosion of the telomeres are related to the death of the cell.

  33. Termination • Thus, extension of telomeres is linked to longer lifespan for the cell. • Enzyme telomerase responsible for extension. • Gene that codes for telomerase is directly linked to the longevity in worms and fruit flies • Cancer cells also contains telomerase.

  34. Proofreading and Correction • Errors occur in DNA replication fairly frequently: the wrong base gets inserted due to the peculiarities of nucleotide chemistry

  35. Proofreading and Correction • DNA polymerase has editing function that removes most of the incorrect bases. • DNA pol detects the absence of hydrogen bonding (when a mismatch occurs), removes the incorrect base and inserts the correct one using the parent strand as a template. • This complex process of replication is known as the replication machine.

  36. Nucleic Acid Modifying Enzymes • Restriction endonucleases (“molecular scalpels”) • DNA polymerases (synthesize DNA) • DNA ligases (join DNA strands by forming a phosphodiester bond) • Kinases (phosphorylation of 5´-terminus of DNA molecule)

  37. Nucleic Acid Modifying Enzymes • Phosphatases (dephosphorylate 5´-terminus of DNA molecule) • Ribonucleases (digest RNA molecule. Example: RNase A) • Deoxyribonucleases (digest DNA molecules)

  38. Restriction Endonucleases (RE) • Found only in microorganisms • Exhibit novel DNA sequence specificities • >2000 distinct restriction enzymes have been identified • Function as homodimer; recognize symmetrical dsDNA (palindromes) • Utilized in the digestion of DNA molecules for hybridization procedures or in the direct identification of mutations

  39. Restriction Enzymes Recognize Palindromes • Palindrome reads the same in both directions • BOB • “Able was I ere I saw Elba.” (Napoleon Bonapart, following his exile from the European continent to the island of Elba) • Sequences directly opposite one another on opposite strands of the ds DNA molecule

  40. Restriction Endonucleases • Recognize specific sequences of 4, 5, or 6 nucleotides • Cut by breaking the phosphodiester bond in both strands • Cutting genomic DNA with a RE results in many fragments of different sizes • The smaller the recognition sequence the larger the number of fragments produced

  41. Cohesive Ends (3´ Overhang) Cohesive Ends (5´ Overhang) Blunt Ends (No Overhang) BamH1 GGATCC CCTAGG KpnI GGTACC CCATGG HaeIII GGCC CCGG Restriction Enzymes

  42. GGATCC CCTAGG BamHI (5’ Overhang) CTCGTG GAGCAG BssSI (5’ Overhang) NNCAGTGNN NNGTCACNN TspRI (3’ Overhang) AGATCT TCTAGA BglII (5’ Overhang) Enzymes Recognizing Non palindromic Sequences Enzymes Generating Compatible Cohesive Ends CCCGGG GGGCCC XmaI (5’ Overhang) GATC CTAG DpnI (Requires methylation) GGCC CCGG HaeIII (Inhibited by methylation) CCCGGG GGGCCC SmaI (Blunt Ends) Methylation-sensitive Enzymes Isoschizomers Restriction Enzymes

  43. Typical Restriction Digestion Reaction • Reactions are composed of DNA template, restriction enzyme, 10X buffer, and distilled water. • The required amounts of these components can be calculated based on the number of specimens to be digested: • L DNA (~10 g/rxn) • L restriction enzyme • L 10X buffer • L distilled water

  44. Modifying Enzymes • DNA ligase • catalyses formation of bonds between 5’-P and 3’-OH groups on backbone of DNA • ligate “blunt end” or “sticky ends” • can repair “nicks” in DNA • DNA polymerase • require primers to extend and copy DNA • all extend 5’3’ by adding on to 3’-OH • make a reverse, complimentary copy

  45. Modifying Enzymes • Nucleases (exonucleases) • cut DNA in non-sequence specific manner • can digest DNA from either 5’3’ or 3’5’ direction • prefer ssDNA • proofreading function of polymerase • Alkaline phosphatase • removes 5’ P, prevents recircularization of plasmids

  46. Modifying Enzymes • DNAse • non-specifically digests DNA, ds or ss • commonly found on most surfaces, including hands • RNAse • many different types, may be specific for ssRNA or RNA/DNA hybrids (RNAse H) • extremely common (especially on hands), very stable

  47. Replication of Chromosomes: Mitosis

  48. Assortment of Chromosomes: Meiosis

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