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Text Box: Chargaff’s Results   Using paper chromatography to separate nitrogen bases Chargaff reported the following: 1.      DNA composition is species specific.  The amounts and ratios of nitrogenous bases vary from one species to another.  This source of molecular diversity made it more credible that DNA is the genetic material. 2.      In every species he studied there was a regularity in base ratios. Chargaff’s  Rule:           A= T    and G=C   Genetic Material Must: 1.      Carry information form generation to generation 2.      Be able to copy itself with each cell division. 3.      Be chemically stable. 4.      Be capable of mutation to allow for genetic variation which leads to evolution by natural selection.    Watson and Crick received a Nobel Prize for their discovery of the double helix.  They built scale models of a double helix that would conform to the X-ray data and known chemistry of DNA.    To see a DNA diagram click here.   DNA is made up of repeating units called nucleotides.  Each nucleotide is made up of 3 chemical groups, a phosphate, deoxyribose (a five carbon sugar), and a nitrogen base.    There are 4 types of nitrogen bases 1)      Adenine (A) 2)      Thymine (T) 3)      Guanine (G) 4)      Cytosine (C) According to the DNA base-pairing rule (Chargaff’s Rule), A and T will always bond together and C and C will always bond together.  The nitrogen bases are linked together by hydrogen bonds.  These bonds are much weaker than all the other bonds in the molecule.        DNA Replication                         DNA is capable of replicating (making an exact copy) of itself.  The DNA molecule ‘unzips’ itself down the middle between the nitrogen bases.  Then nucleotides from the cell attach to the exposed bases.  For example, if thymine is exposed then adenine binds to it.  If cytosine is expose then guanine will bond to it.              Eventually both sides of the ‘unzipped’ DNA molecule will end up with the same nucleotides as were in the original, so two identical DNA molecules result as replication is completed.      The general mechanism of DNA replication is conceptually simple but the actual process is complicated.  The helical molecule must untwist while it copies its two antiparallel strands simultaneously.  This requires over a dozen enzymes and other proteins.  It is also extremely rapid.  In prokaryotes up to 500 nucleotides are added per second.  It takes only a few hours to copy the 6 billion bases of a single human cell.  DNA replication is also accurate.  Only about one in a billion nucleotides is incorrectly paired.    To see Meselson Stahl Experiment Diagram click here.   DNA replication begins at special sites called origins of replication that have a specific sequence of nucleotides.  Specific proteins required to initiate replication bind to each origin.  The DNA double helix opens at the origin and replication forks spread in both directions away from the central initiation point creating a replication bubble. Bacterial or viral DNA molecules have only one replication origin.  Eukaryotic chromosomes have hundreds or thousands of replication origins.  The many replication bubbles eventually merge forming two continuous DNA molecules.    Replication Forks: Y shaped regions of replicating DNA where new strands are formed.   Enzymes called DNA polymerases catalyze synthesis of a new DNA strand.  According to the base pairing rules, new nucleotides align themselves along the templates of the old DNA strands.  DNA polymerase links the nucleotides to the growing strand.  These strands grow in the 5’ to 3’ direction since new nucleotides are added only to the 3’ end of the growing strand.    DNA polymerase:  Enzyme that binds nucleotides to the growing DNA strand in the 5’ to 3’ direction.  This enzyme functions on both the leading and lagging strand of DNA.    DNA ligase: Enzyme that ligates (binds together) the Okazaki fragments on the lagging strand of DNA. It is responsible for joining the phosphate from one Okazaki fragment to the sugar on the adjacent Okazaki fragment.   DNA replication is highly accurate, but this accuracy is not solely the result of base-pairing specificity.  Enzymes proofread DNA during its replication and repair damage to existing DNA.  Initial pairing errors occur at a frequency of about one in ten thousand, while errors in a complete DNA molecule are only about one in one billion.  DNA can be repaired as it is being synthesized (mismatched repair) or after accidental changes in existing DNA (excision repair0.    Mismatch Repair – Corrects bases when DNA is synthesized.   In bacteria, DNA polymerase proofreads each newly added nucleotide against its template.  If polymerase detects and incorrectly paired nucleotide, the enzyme removes and replaces it before continuing with synthesis.  In eukaryotes additional proteins as well as polymerase participate in mismatch repair.  A hereditary defect in one of these proteins has been associated with a form of colon cancer.  Apparently, DNA errors accumulate in the absence of adequate proofreading.    Excision Repair – Corrects accidental changes that exist in already existing DNA.    Accidental changes in DNA (mutations) can result from exposure to reactive chemicals, radioactivity, X-rays, and ultraviolet light.  There are more than fifty different types of DNA repair enzymes that repair damage.  For example in excision repair the damaged segment is excised by one repair enzyme and the remaining gap is filled in by base-pairing nucleotides with the undamaged strand.  DNA polymerase and DNA ligase are enzymes that catalyze the filling in process.