➢ Chapter 16 ➢ ➢ Griffith tried to develop a vaccine against pneumonia ○ Two strains: pathogenic and non-pathogenic ○ When the pathogenic bacteria were killed with heat and mixed with the nonpathogenic strains,some of the living cells became pathogenic ■ Some chemical component of the dead pathogenic cells caused this heritable change, known as transformation ➢ Transformation: a change in genotype and phenotype due to the assimilation of external DNA by a cell ➢ Hershey and Chase proved that DNA was genetic material, not proteins ○ After Griffith’s experiment, scientists were skeptical of what genetic material was because viruses have a protective coat that is often made of protein ○ Used a radioactive isotope of sulfur to tag proteins in one batch and radioactive isotope of phosphorus to tag DNA in a second batch ○ Tested the samples shortly after infection and found that the phage DNA entered the host cells but not the phage protein ○ Cells released phages that contained some radioactive phosphorus DNA inside the cell played an ongoing role during the infection process ➢ Chargaff discovered that ration of A:T and C:G is approximated 1 because the base pairs appear in nearly equal amounts ○ Base composition (percentage of each base) different among different species DNA contributes to genetic diversity ➢ Double Helix: presence of two strands in DNA ➢ Antiparallel: subunits of DNA run in opposite directions ➢ Adenine pairs with thymine and cytosine pairs with guanine ○ Thymine is replaced with uracil in RNA ○ Purines: adenine and guanine ■ Two organic rings ○ Pyrimidines: cytosine and thymine ■ Single organic ring ○ Difference in sizes of pyrimidines and purines accounts for the asymmetry in DNA o Weak interactions known as hydrogen bonds ➢ Semiconservative Model: when a double helix replicates, each of the two daughter molecules will have one old strand from the parental molecule and one new strand ➢ Origins of Replication: short stretches of DNA having a specific sequence of nucleotides ○ Proteins that initiate DNA replication recognize this sequence and attach to the DNA, separating the two strands and opening up a replication “bubble” ○ Having hundreds of origins of replications starts multiple replication bubbles that speed up the process ➢ Replication Fork: Y-shaped region where the parental strands of DNA are being unwound at the end of reach replication bubble ○ Helicases: enzymes that untwist the double helix, separating the two parental strands and making them available as template strands ○ Single Strand Binding Proteins: bind to the unpaired DNA strands, keeping them from re-pairing ○ Topoisomerase: relieves strain caused by the untwisting of the double helix by breaking, swiveling, and rejoining DNA strands ➢ Unwound sections of parental DNA strands are now available to serve as templates for the synthesis of new complementary DNA strands. ➢ Enzymes that synthesize DNA cannot initiate the synthesis ○ Primer: DNA synthesis is actually a short stretch of this RNA strand o Primase: enzyme that synthesis primer ■ Starts a complementary RNA chain from a single RNA nucleotide, adding more ■ 5-10 nucleotides long ■ New DNA strand will start from the 3’ end of the RNA primer ➢ DNA Polymerases: catalyze the synthesis of new DNA by adding nucleotides to a preexisting chain ○ Two main ones are DNA polymerase III and DNA polymerase I o Most require a primer and DNA template strand ➢ Antiparallel Elongation ○ DNA polymerases can add nucleotides only to the free 3’ end of a primer or growing DNA strand ■ New DNA strand only elongations from the 5’ 3’ direction ■ Leading Strand ○ To elongation the other new strand of DNA in 5’ 3’, DNA polymerase III must work along the other template strand in the direction away from the replication fork ■ Lagging Strand ■ Synthesized discontinuously as a series of fragments known as Okazaki fragments ■ DNA ligase joins the Okazaki fragments ➢ Summary of DNA Replication: 1. Helicase unwinds the parental double helix 2. Molecules of single-strand binding protein stabilize the unwound template strands 3. The leading strand is synthesized continuously in the 5’ to 3’ direction by DNA polymerase III 4. Primase begins synthesis of the RNA primer for the fifth Okazaki fragment 5. DNA polymerase III is completing synthesis of the 4th fragment and when it reaches the RNA primer on fragment 3, it will detach and begin adding nucleotides to the 3’ end of the fragment 5 primer in the replication fork 6. DNA polymerase I removes the primer from the 5’ end of fragment 2, replacing it with DNA nucleotides adding one by one to the 3’ end of fragment 3. After the last addition, the backbone is left with a free 3’ end. 7. DNA ligase joins the 3’ end of fragment 2 to the 5’ end of fragment 1 ➢ Mismatch Pair: other enzymes remove and replace incorrectly paired nucleotides that have resulted form replication errors ➢ Nuclease: DNA-cutting enzyme ○ When there is an error in replication, the incorrect part is cut out by nuclease and replaced with nucleotides using the undamaged strand as a template ■ Ex: nucleotide excision repair ➢ Once a mismatched nucleotide pair is replicated, the sequence change is permanent in the daughter molecule and in any subsequent copies ○ Mutation: permanent change in DNA ■ Can change the phenotype of an organism ➢ Telomeres: eukaryotic chromosomal DNA molecules have these nucleotide sequences at their ends ○ Do not have genes, but multiple repetitions of one short nucleotide sequence ➢ Two protective functions: ○ Specific proteins associated with telomeric DNA prevent the staggered ends of the daughter molecule from activating the cell’s systems for monitoring DNA damage ○ Telomeric DNA acts as a buffer zone that provides some protection against the organism’s genes shortening (do not prevent the erosion, simply postpone it) ➢ Become shorter during every round of replication ○ Telomerase: enzyme that catalyzes the lengthening of telomeres in eukaryotic germ cells to restore their original length ○ Cells from large tumors have unusually short telomeres because they have undergone many cell divisions ○ Chromatin: complex of DNA and protein (histones) fits into the nucleus through a multilevel parking system Questions 1. C 2. C 3. B 4. D 5. A 6. D 7. B 8. A 9. histones have a positive charge in their N-terminal end which allows them to tightly bind to the negatively charged DNA. Based on this, it would make sense that the proteins that bind to DNA in E. coli would also have a positive charge, similar to histones in eukaryotes. This positive charge helps facilitate the binding between the proteins and the DNA.
