Question

2a-1pt) Arthur Kornberg and his colleagues incubated an extract of E. coli with a mixture of dATP, dTTP, dGTP, and dCTP. Only the dTTP was labeled with 32P in the a-phosphate group. After incubation, the mixture was treated with trichloroacetic acid to precipitate DNA, but not the dNTPs. The precipitate was collected, and the extent of [32P] dTMP incorporated into DNA was determined. If any one of the dNTPs were omitted from the incubation mixture, would radioactivity be found in the precipitate? Pick the answer choice.

i) Yes. In the absence of any one dNTP, the polymerase would incorporate the alternative purine or pyrimidine (e.g., insert thymidine if cytosine was missing). Thus, the [32P]dTMPs would be found in the precipitate.

ii) No. In the absence of any one dNTP, the polymerase would stop incorporating the other three dNTPs as soon as it encountered a template residue that should pair with the missing dNTP, and incorporation of 32P would be undetectable.

2b-1pt) In the same assay described above would radioactivity be found in the precipitate if the 32P label was in the β- or γ-phosphate position? Why? (Use the answers options above as a template for how to answer question. Use of correct terminology as it pertains to chemistry of DNA synthesis is key)

Answer 1

2a. Option ii is correct.

If any of the dNTPs is omitted in the reaction mixture, DNA polymerase would not be able to complete DNA replication. So, the precipitate would not contain a DNA strand with labeled dTMP.

2b. dNTPs serve a dual purpose during replication. First, they serve as the reactants for DNA replication. Second, the phosphodiester bond cleavage between beta and gamma-phosphate groups provides the energy required for the incorporation of a new nucleotide into the strand.

Existing strand + dNTP ------> Existing strand-dNMP + PPi

If the label is present on beta or gamma phosphate, it would not be incorporated into the DNA as the pyrophosphate is released during the reaction.