BMB 462 Lecture 22 Outline of Last Lecture I Mutations in DNA II Beneficial Methylation Begin Unit on Chromosome Structure III IV V VI VII Composition of the Genome Supercoiling in Chromosomes Function of Topoisomerases Nucleosome Formation and other Chromosome Structures Maintenance of Chromosomal Structure Outline of Current Lecture I Models for DNA Replication II Properties of DNA Replication III Requirements for in vitro synthesis IV Comparing the DNA polymerases Current Lecture Concepts to remembers from previous courses lectures I Models for DNA Replication a Semi conservative i The Watson Crick model ii Each daughter chromatid has one original strand of DNA and one new strand of DNA b Conservative i The DNA is used as a template to make a new copy but then the original strands pair up again and the new strands pair to make an entirely new helix c Dispersive These notes represent a detailed interpretation of the professor s lecture GradeBuddy is best used as a supplement to your own notes not as a substitute i Each strand is composed of bits and pieces of new DNA and old DNA that is combined together II Properties of DNA Replication a The Meselson Stahl experiment determining that DNA replication is semiconservative i To determine the model for replication bacteria were grown on 15N media When the N isotope was incorporated into the nucleotides all the DNA strands were heavy and appeared at the bottom of the centrifuge tube ii In the second generation they switched to the normal 14N media The DNA appeared in the center of the tube showing hybrid helices heavy and normal isotopes were incorporated iii In the next generation there were light DNA and hybrid DNA b Bidirectional Replication Visualized by Cairn via DNA with 3H labeled thymidine i When the new DNA nucleotides were labeled with heavy isotopes 2 areas of heavy labeled DNA were apparent The rest of the chromosome had light DNA 1 This indicates that there are 2 replication forks so replication is bidirectional c Origin of Replication Inman s denaturation mapping i Replication doesn t start randomly in the DNA There are unique sequences that replication originates from ii They were able to landmark AT rich regions and then would isolate replicating DNA from a phage This showed that replication starts at a unique origin d DNA replication progresses 5 to 3 i DNA is read from 5 to 3 new bases are always added to the 3 end of the new strand 1 This causes problems with the lagging strand because it runs in the opposite direction ii In the leading strand the strand is read in the correct direction so new bases can be added continuously e Semi discontinuous Replication Okazaki Fragments i In the experiment the bases were labeled with 3H thymidine and then the contents were centrifuged Researchers were able to determine the lagging strand replicated in fragments instead of continuously III Minimum Requirements for in vitro synthesis a Nucleotides the building blocks They provide energy as well b DNA polymerase c DNA primers the primers provide the 3 OH so that the replicating DNA has a base to build off of and determine where replication starts d A DNA template a strand of DNA that gets heated to denature Then cool it so the primers can anneal so that new bases can be added e Buffer and Mg2 IV Comparing the DNA polymerases a General Properties i Polymerases involved in replication are DNA dependent DNA polymerases they require a DNA template ii All polymerases undergo 5 to 3 synthesis so they require an attached primer with 3 OH 1 When the new dNTP is added the phosphate attacks the 3 OH and a new bond is formed iii Processive they can add more than 1 nucleotide before they fall off the DNA some can add 500 000 nucleotides iv Accurate Polymerases are highly accurate at synthesizing DNA 1 i e DNA polymerase III makes 1 error in 1x104 to 105 nucleotides added DNA polymerase III also has proofreading abilities that increase accuracy to about 1 error in 106 107 v Mismatch repair further increases accuracy to 109 1010 vi The E coli genome which is 4 6 x106 base pairs experiences 1 error every 100 replications 1 With mismatch repair that decreases to 1 every 1000 to 10 000 replications vii E coli adds 1000nucleotides sec Eukaryotes add bases slower approximately 500nucleotides sec b Mechanism for DNA synthesis with Polymerases i The primer is complementary to the template strand which is how the polymerase knows where to attach and start replication 1 The new nucleotide also has to be complementary to the template strand ii The phosphate groups are coordinated to Mg2 in the active site of the polymerase iii The incoming dNTP is attacked at the phosphate by the 3 OH of the growing DNA chain 1 The phospho anhydride bond is broken and pyrophosphate leaves iv A new phosphodiester bond is created c Base Selection i DNA polymerase I has 2 active sites one that selects the new nucleotide and one that has proofreading abilities in the exonuclease active site 1 Replication has to be very accurate so the cell and more specifically the polymerase must take extra precautions to be sure the correct base is added ii The exonuclease active site recognizes the shape and size of the correct base pair 1 In an incorrect pairing the exonuclease breaks the bond and expels the incorrect nucleotide so that the correct one can be added in iii When incorrect pairing occurs base pair stacking is impaired because the strand becomes less stable iv With base selection alone there is 1 error every 104 to 105 nucleotides added d DNA Proofreading 3 to 5 Exonuclease Activity i First a bond is created in the polymerase active site 1 In incorrect pairing the base pair gets a weird shape The polymerase recognizes this and then moves the base pair to the exonuclease active site where the bond is broken ii Recognizing the mismatch the polymerase slides back so that the nucleotide is in the exonuclease site The phosphodiester bond is broken 1 It s not a real reversal of the polymerase reaction because that incorrect nucleotide is lost a A dNMP is removed instead of a dNTP therefor energy is lost in this process but it is more crucial that the DNA sequence is accurately preserved e Comparing DNA polymerases I II and III E coli i Polymerase III is the major replicative polymerase ii Polymerase I is involved in replication but also in repair and recombination 1 5 3 exonuclease in polymerase I removes the primer on lagging strand iii Polymerase II is only involved in
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