To study molecular mechanism of DNA replication you incubate soluble E. coli ext

Question

To study molecular mechanism of DNA replication you incubate soluble E. coli extracts with a mixture of dATP, dTTP, dGTP and dCTP all of which are labeled with a phosphate group. After awhile the incubation mixture was treated with trichloroacetic acid, which precipitate the DNA but not the nucleotide or very short oligonucleotides. The precipitate was collected and the extent of the radioactively label nucleotide incorporation into the DNA was determined 1) If any one of the four nucleotides is omitted from the incubation mixture, would you detect radioactivity in the precipitate? Explain 2) Would 32P be incorporated into the DNA if only dTTP is labeled? Explain 3) Would radioactivity be found in the precipitate if 32P be labeled the Theta and lambda rather than the alpha phosphate if the deoxyribonucleotides? Explain?

Explanation / Answer

DNA templating is the process in which the nucleotide sequence of a DNA strand (or selected portions of a DNA strand) is copied by complementary base-pairing (A with T, and G with C) into a complementary DNA sequence (Figure 5-2). This process entails the recognition of each nucleotide in the DNA template strand by a free (unpolymerized) complementary nucleotide, and it requires that the two strands of the DNA helix be separated. This separation allows the hydrogen-bond donor and acceptor groups on each DNA base to become exposed for base-pairing with the appropriate incoming free nucleotide, aligning it for its enzyme-catalyzed polymerization into a new DNA chain. If the DNA polymerase did nothing special when a mispairing occurred between an incoming deoxyribonucleoside triphosphate and the DNA template, the wrong nucleotide would often be incorporated into the new DNA chain, producing frequent mutations. The high fidelity of DNA replication, however, depends not only on complementary base-pairing but also on several “proofreading” mechanisms that act sequentially to correct any initial mispairing that might have occurred. The first proofreading step is carried out by the DNA polymerase, and it occurs just before a new nucleotide is added to the growing chain. Our knowledge of this mechanism comes from studies of several different DNA polymerases, including one produced by a bacterial virus, T7, that replicates inside E. coli. The correct nucleotide has a higher affinity for the moving polymerase than does the incorrect nucleotide, because only the correct nucleotide can correctly base-pair with the template. Moreover, after nucleotide binding, but before the nucleotide is covalently added to the growing chain, the enzyme must undergo a conformational change. An incorrectly bound nucleotide is more likely to dissociate during this step than the correct one. This step therefore allows the polymerase to “double-check” the exact base-pair geometry before it catalyzes the addition of the nucleotide. The next error-correcting reaction, known as exonucleolytic proofreading, takes place immediately after those rare instances in which an incorrect nucleotide is covalently added to the growing chain. DNA polymerase enzymes cannot begin a new polynucleotide chain by linking two nucleoside triphosphates together. Instead, they absolutely require a base-paired 3'-OH end of a primer strand on which to add further nucleotides (see Figure 5-4). Those DNA molecules with a mismatched (improperly base-paired) nucleotide at the 3'-OH end of the primer strand are not effective as templates because the polymerase cannot extend such a strand. DNA polymerase molecules deal with such a mismatched primer strand by means of a separate catalytic site (either in a separate subunit or in a separate domain of the polymerase molecule, depending on the polymerase). This 3'-to-5' proofreading exonuclease clips off any unpaired residues at the primer terminus, continuing until enough nucleotides have been removed to regenerate a base-paired 3'-OH terminus that can prime DNA synthesis. In this way, DNA polymerase functions as a “self-correcting” enzyme that removes its own polymerization errors as it moves along the DNA.

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