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Unformatted text preview: \]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. 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 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). 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 . Such a high means that the DNA synthesis reaction is not spontaneously reversible to any great degree. 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. DNA pol III Single origin in the bacteria. Multiple origins in Domain Eukarya. The E. coli model. Helicase ATP powered from now on we need to know energy source sometimes it will be gtp for helicase rna helicase runs along breaks hydrogen bonds and opens the dna Single-stranded binding proteins. Not enzymes Supercoiling relaxed by topoisomerases both in euchara and ccDNA Primase Primase and other proteins form functional primosome. Rna polymerase can start without a primer template junction another observation congruent with rna as being first All dna synthesis begins 5’ new to 3’ new energy comes in on five Many times an enzyme will be a complex of several proteins Core protein helps reaction other proteins hold everything together holo enzyme...
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