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Ch 7 Notes Part II - (dATP,dTTP,etc...

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The Chemistry of DNA synthesis Synthesis of DNA requires two substrates: The four deoxynucleoside triphosphates (dATP, dTTP, etc.) And a primer: template junction Deoxynucleoside triphosphates have three phosphoryl groups named  alpha, beta and gamma . Alpha is the innermost group. The primer: template junction is an arrangement of ssDNA and dsDNA. The primer must have an exposed 3’OH in order for synthesis to proceed. The primer is complementary too and shorter than the template. In both DNA and RNA synthesis it is the  3’ end  of the molecule that is extended by the addition of  new monomers. (5’ to 3’ because energy before the process) The formal substrate for DNA synthesis is the primer since it is chemically changed by DNA  synthesis. The template only provides the information for the ordering of the new monomers. The phosphodiester bond between the new monomer and the polymer is formed when the  hydroxyl group at the 3’ end of the primer attacks the  α -phosphoryl group  of the new monomer. The leaving group for the reaction (breaking of phosophate dimmer) is a  pyrophosphate , which  arises from the release of the  β -phosphoryl and  γ -phosphoryl groups covalently bonded together. Note it is a phosphate dimer. The free energy for this reaction is rather small ( G= -3.5 kcal/mole). Memorize. The driving force for the polymerization reaction is provided by the immediate hydrolysis of  pyrophosphate into two phosphate groups. See reactions pp193, 184 MBG HO. This reaction is catalyzed by the enzyme,  Pyrophosphatase . The overall  G = -7 kcal/mole for the coupled process which corresponds to a K eq  of about 10 5 (Memorize) Such a high K eq  means that the DNA synthesis reaction is not spontaneously reversible to any  great degree.
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An overview of the enzymes operating at the replication fork Enzyme DNA polymerase. DNA polymerase I (DNA pol I), the first DNA polymerase discovered, was discovered by Arthur  Kornberg in  E. coli  in the late 1950’s. (numbered in order of discovery) DNA pol III Single origin in the bacteria. Multiple origins in Domain Eukarya.   Pulse- you give the cell some radioactive form that participates in a particular process that you  want to look at Chase- gives you some of the same type of molecule but not radioactive The  E. coli  model. Helicase ATP powered  Single-stranded binding proteins (not an enzyme!)- the job is to keep single stranded DNA from  renanniling  Supercoiling relaxed by topoisomerases Primase Primase and other proteins form functional primosome.
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