DNA Replication
Chapter 16: DNA as Genetic Material Genes are on Chromosomes T.H. Morgan o Working with Drosophila (fruit flies) o Genes are on chromosomes o But is it the protein or the DNA of the chromosomes that are the genes? Through 1940 proteins were thought to be genetic material Why? The Transforming Factor Frederick Griffith o Streptococcus pneumonia bacteria Was working to find cure for pneumonia. o Harmless live bacteria mixed with heat killed infectious bacteria causes disease in mice. o Substance passed from dead bacteria to live bacteria = Transforming Factor. Transformation: change in genotype & phenotype due to assimilation of external DNA by a cell. DNA is the Transforming Factor Avery, McCarty & MacLeod Purified both DNA & proteins from Streptococcus pneumonia bacteria. o Which will transform non-pathogenic bacteria? Injected protein into bacteria. o No effect Injected DNA into bacteria o Transformed harmless bacteria into virulent bacteria. What is the conclusion? 1
Confirmation of DNA Hershey & Chase o Classic blender experiment o Worked with bacteriophage Viruses that infect bacteria o Grew phage viruses in 2 media, radioactively labeled with either: 35S in their proteins 32P in their DNA o Infected bacteria with labeled phages. Chargaff DNA composition: Chargaff s rules o Varies from species to species o Amounts of 4 bases not equal o Bases present in characteristic ratio Humans: A=30.9% T=29.4% G=19.9% C=19.8% Structure of DNA Watson & Crick o Developed double helix model of DNA Other scientists working on question: o Linus Pauling o Maurice Wilkins o Rosalind Franklin Anti-parallel strands Phosphate to sugar bond involves carbons in 3 & 5 positions DNA molecule has direction Complementary strand runs in opposite direction Bonding in DNA Base pairing in DNA Purines o Adenine o Guanine Pyrimidines o Thymine o Cytosine Pairing o A : T o C : G 2
Copying DNA Replication of DNA o Base pairing allows each strand to serve as a pattern for a new strand. Models of DNA Replication o Conservative Model o Semiconservative Model o Dispersive Model Semi-conservative Replication Meselson & Stahl o Labeled nucleotides of parent DNA strands with heavy nitrogen = 15 N o Labeled new nucleotides with lighter isotope = 14 N o Replicated strands were found to be half 15 N & half 14 N DNA Replication Large team of enzymes coordinates replication. 3
DNA Enzymes Helicase o Opens DNA helix enabling replication DNA Polymerase III o Adds nucleotides only to 3 end. o Nucleoside-P-P-P links to sugar-p backbone. o Losing 2-P provides energy for bonding. Leading & Lagging strands Leading strand o Continuous synthesis Lagging strand o Okazaki fragments Joined by ligase o spot welder Okazaki Fragments Priming DNA synthesis DNA polymerase can only extend an existing DNA molecule; it cannot start a new one. o Short RNA primer is built first on parent DNA strand by primase. o RNA primer later removed by DNA polymerase I 4
Replication Enzymes DNA polymerases DNA polymerase I o 20 bases/second o Editing, repair & primer removal DNA polymerase III o 1000 bases/second o Main DNA building enzyme Editing & proofreading DNA At 1000 bases/second, lots of typos! DNA polymerase I excises mismatched bases o Reduces error rate from 1 in 10,000 to 1 in 100 million bases Fast & accurate! It takes E. coli <1 hour to copy 5 million base pairs in its single chromosome & divide to form 2 identical daughter cells Human cell copies its 6 billion bases & divide into daughter cells in only few hours o Remarkably accurate o Only ~1 error per 100 million bases o ~30 errors per cell cycle Problems in the end Ends of chromosomes are eroded with each replication Telomeres Expendable, non-coding sequences at ends of DNA. o Short sequence of bases repeated 1000s times. o TTAGGG in humans Telomerase enzyme in certain cells. 5