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Molecular Basis of Inheritance Chapter 14 •DNA Studies • Frederick Griffith – 1928 • Streptococcus •2 pneumoniae strains – pathogenic & harmless • Killed pathogenic mixed with living harmless living cells converted to pathogenic offspring inherited pathogenic •DNA Studies • Called • process transformation Substance transforming was DNA •DNA Studies • Hershey & Chase – 1952 • Studied bacteriophages (viruses that infect bacteria) • Virus – mostly DNA & protein • Through radioactive isotopes, studied T2 (virus that infects E. coli in mammalian intestine) •DNA Studies • Each separately added to E. coli: • Radioactive sulfur for proteins • Radioactive phosphorus for DNA • Culture allowed to grow • Bacteria broken from virus & studied – radioactivity in bacteria with phosphorus • Result – DNA entered bacteria, not protein •DNA Studies • Erwin Chargaff – determined A-T and G-C •DNA Structure • James Watson & Francis Crick •DNA Structure • Through use of Rosalind Franklin’s X-ray diffraction photograph, W&C determined double helix structure •DNA Structure • Sugar-phosphate • Nitrogenous backbone bases on inside (10 per turn of helix) • Bond purine to pyrimidine •A has 2 H bonds with T only •G has 3 H bonds with C only •DNA Replication • DNA untwists & unzips • Complementary base pairs free in cytosol bond appropriately • New strands re-zip and re-twist •DNA Replication • Result – Two daughter DNA molecules • One parent strand • One new strand • Called the semi-conservative model •Origins of Replication • Where DNA replication begins • Has specific nucleotide sequence • Proteins recognize this at these sites help separate DNA (open replication “bubble”) •Origins of Replication • Can be hundreds of bubbles • Extending from bubble – replication fork (where new strands are elongating) • Replication in both directions until bubbles fuse •DNA Elongation • DNA Polymerases – enzymes aiding elongation at a replication fork •Strand Arrangement • Opposite sides of backbone run antiparallel (upside down) to each other • 5’ end – phosphate • 3’ end – hydroxyl group • Phosphates connect from 5’ C of one sugar to 3’ C on next sugar •Strand Arrangement • FYI…find nitrogenous base…that is 1’C… count clockwise to find others • New nucleotides are added ALWAYS from 5’ end of the new DNA to 3’ end (3’-5’ of old strand) •Strand Arrangement • When DNA unzips, the 3’-5’ strand can fill in easily – leading strand • Copies toward replication fork • Helped by DNA polymerase •Strand Arrangement • The 5’-3’ strand fills in with pieces – lagging strand • Pieces called Okazaki fragments • DNA ligase helps to join sugars & phosphates together •How Does It Start? • When replication starts, new chain begins with a primer • Section of RNA • Primase joins approx 10 RNA nucleotides together to start replication • Later replaced by DNA with DNA polymerase help •How Does It Start? • One primer for leading strand • Each fragment of lagging strand is primed & then replaced by DNA •Other help • Helicase – enzyme untwists DNA • Single-strand binding proteins – keep DNA apart during process • Overview of DNA Replication •Proofreading • Mismatch repair • Polymerase matches new nucleotide to parent strand will remove if incorrect • Each cell monitors DNA for new changes due to cell error or envi. • Nucleotide Excision Repair • New error found a nuclease cuts it out polymerase & ligase fill in proper pieces •Last fix • Once the last RNA primer comes off, there is a gap that needs to be fixed •Last Fix • Nucleotides can only add to the 3’ end of a preexisting polynucleotide • No way to complete 5’ end • Over time, DNA would progressively shorten – problem • Solution – telomerase •Last Fix • End of DNA – telomeres • Repetitive expendable (non-coding) nucleotide sequence • Protect • Will major shortening of DNA shorten somewhat over time • Telomerase will help lengthen •Last Fix • Has RNA on it • Serves as template to extend telomere at 3’ end of the telomere • Telomeres • Telomeres and Cancer