To study the molecular mechanism of DNA replication, you incubated soluble E. co

Question

To study the molecular mechanism of DNA replication, you incubated soluble E. coli extracts with a mixture of dATP, dTTP, dGTP, and dCTP, all of which are labeled with the a-phosphate group. After a while, 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. If any one of the four nucleotides is omitted from the incubation mixture, would you detect radioactivity in the precipitate? Explain Would 32P be incorporated into the DNA if only dTTP is labeled? Explain Would radioactivity be found in the precipitate if 32P labeled the b and g rather than the a phosphate of the deoxyribonucleotides? Explain.

Explanation / Answer

a) No.

Incorporation of 32P into DNA results from the synthesis of new DNA,

which requires the presence of all four nucleotide precursors.

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(b) Yes.

Although all four nucleotide precursors must be present for DNA synthesis, only

one of them has to be radioactive in order for radioactivity to appear in the new DNA

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c) No.

No radioactivity would be incorporated if the 32P label were not at the a phosphate

because DNA polymerase, which catalyzes this reaction, cleaves off pyrophosphate—that

is, the b and g phosphate groups

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