Nucleic Acid Structure and Replication - DNA - Synan AbuQamar PDF

NUCLEIC ACID STRUCTURE AND REPLICATION Biochemical Identification of the genetic Material Nucleic Acid Structure DNA Replication By: Synan AbuQamar Genetic material Criteria: Contain the information necessary to construct an entire organism Pass from parent to offspring and from...

NUCLEIC ACID STRUCTURE AND REPLICATION Biochemical Identification of the genetic Material Nucleic Acid Structure DNA Replication By: Synan AbuQamar Genetic material Criteria: Contain the information necessary to construct an entire organism Pass from parent to offspring and from cell to cell during cell division (transmission) Be accurately copied (replication) Account for the known variation within and between species 2  Genetic Material – DNA or Protein? The answer! The Griffith bacterial transformation Experiment: Bacteria Streptococus pneumoniae Injection into mice Experiment 1: 2 strains were injected: S strain: produce Shiny and smooth colonies. S strain is encapsulated R Strain: produce Rough appearance colonies Observations: S strain injected to mice: mice died (virulent), Virulence due to the capsule R strain injected to mice: mice did not die (non-virulent)  Observations: Living S strain were recovered from body Substance necessary for virulence passed from dead S strain to living R strain Bacteria R strain bacteria was transformed Conclusion: The transforming substance passed was genetic material. Hershey and Chase 1952, studying T2 virus infecting Escherichia coli  Bacteriophage (phage) Phage coat made entirely of protein DNA found inside capsid 5  The Hershey-Chase Experiment Chemical differences between DNA and Protein: - Phosphate is present in DNA - Sulfate is present in Protein Hershey and Chase used radioactive 32P to label the DNA of the phage and, radioactive 35S to label protein of the phage. Conclusion: Viral DNA (not protein) enters the host. Therefore, the DNA is the genetic material Deoxyribonucleic 11.2 Structure of DNA Nitrogenous bases Acid (DNA) 4 types of nucleotides, Each containing: - Nitrogen containing Base - Phosphate group (Phosphoric Acid) - Pentose Sugar Double Helix: 2 strands DNA contains: Two with purine bases (double ring) Pentoses - Adenine (A) Phosphoric acid - Guanine (G) Two with pyrimidine bases (single ring) - Thymine (T) - Cytosine (C)  Conventional numbering system Sugar carbons 1’ to 5’ Base attached to 1’ Phosphate attached to 5’ 8  Levels of DNA structure 1. Nucleotides are the building blocks of DNA (and RNA). 2. A strand of DNA (or RNA) 3. Two strands form a double helix. 4. In living cells, DNA is associated with an array of different proteins to form chromosomes. 5. A genome is the complete complement of an organism’s genetic material. Nucleotides covalently bonded Phosphodiester bond – phosphate group links 2 sugars Phosphates and sugars from backbone Bases project from backbone Directionality- 5’ to 3’ For example: 5’ – TACG – 3’ 9 Solving DNA structure 1953, J. Watson and F. Crick, with M. Wilkins, proposed the structure of the DNA double helix Watson and Crick used L. Pauling’s method of working out protein structures using simple ball- and-stick models Rosalind Franklin’s X-ray diffraction results provided crucial information E. Chargoff analyzed base composition of DNA that also provided important information 10  Watson and Crick put together these pieces of information Found ball-and-stick model consistent with data Watson and Crick awarded Nobel Prize in 1962 Rosalind Franklin had died and the Nobel is not awarded posthumously 11  DNA is  Double stranded  Helical  Sugar-phosphate backbone  Bases on the inside  Stabilized by H-bonding  Base pairs with specific pairing 12  AT/GC or Chargoff’s rule  A pairs with T  G pairs with C Keeps with consistent 10 base pairs per turn 2 DNA strands are complementary  5’ – GCGGATTT – 3’  3’ – CGCCTAAA – 5’ 2 strands are antiparallel  One strand 5’ to 3’  Other stand 3’ to 5’ 13  Chargaff’s Rules Edwin Chargaff, in 1951, demonstrated that the 4 nucleotides (A, C, G & T) are not equally present in the DNA and that the ratio varies a lot between species. In each species, the amount of A=T and the amount of G=C. The percentage of A+G equals 50% and the percentage of T+C equals 50%  11.3 DNA Replication 3 possible schemes for DNA replication Meselson-Stahl experiment, 1958 Each old strand of DNA serves as a template for a new strand. Semiconservative - One old strand is conserved in each daughter molecule.  During replication 2 parental strands separate and serve as template strands New nucleotides must obey the AT/GC rule (Chargaff’s Rules) End result 2 new double helices with same base sequence as original 16  Origin of replication  Site of start point for replication Bidirectional replication  Replication proceeds outward in opposite directions Bacteria have a single origin Eukaryotes require multiple origins 17  Origin of replication provides an opening called a replication bubble that forms two replication forks DNA replication occurs near the fork Synthesis begins with a primer Proceeds 5’ to 3’ Leading strand made in direction fork is moving  Synthesized as one long continuous molecule Lagging strand made as Okazaki fragments that have to be connected later 18  DNA helicase  Binds to DNA and travels 5’ to 3’ using ATP to separate strand and move fork forward Unwinding or “unzipping” H bonds break DNA topoisomerase  Relives additional coiling ahead of replication fork Single-strand binding proteins  Keep parental strands open to act as templates 19  DNA polymerase  Covalently links nucleotides Complementary base Pairing: Complementary nucleotides are used Joining: Formation of new strands – daugther DNA has 1 old and 1 new strand Deoxynuceloside triphosphates  Free nucleotides with 3 phosphate groups  Breaking covalent bond to release pyrophosphate (2 phosphate groups) provides energy to connect adjacent nucleotides 20  DNA polymerase has 2 enzymatic features to explain leading and lagging strands 1. DNA polymerase unable to begin DNA synthesis on a bare template strand DNA primase must make a short RNA primer  RNA primer will be removed and replaced with DNA later 2. DNA polymerase can only work 5’ to 3’ (directional synthesis) 21  In the leading strand  DNA primase makes one RNA primer  DNA polymerase attaches nucleotides in a 5’ to 3’ direction as it slides forward In the lagging strand  DNA synthesized 5’ to 3’ but in a direction away from the fork  Okazaki fragments made as a short RNA primer made by DNA primase at the 5’ end and then DNA laid down by DNA polymerase  RNA primers will be removed by DNA polymerase and filled in with DNA  DNA ligase will join adjacent DNA fragments 22 DNA replication is very accurate 3 reasons 1. H-bonding between A and T or G and C more stable than mismatches 2. Active site of DNA polymerase unlikely to form bonds if pairs mismatched 3. DNA polymerase removes mismatched pairs “Proofreading” results in DNA polymerase backing up and digesting linkages Other DNA repair enzymes 23