How did Kornberg assess the fidelity of DNA polymerase I in copying a DNA template?

Verified step by step guidance

1. Understand that fidelity in DNA polymerase refers to the enzyme's accuracy in incorporating the correct nucleotides complementary to the DNA template strand during DNA synthesis.
2. Recognize that Kornberg assessed fidelity by measuring the frequency of errors (mutations) introduced by DNA polymerase I when copying a known DNA template sequence.
3. Kornberg used an experimental setup where DNA polymerase I was allowed to synthesize a new DNA strand using a single-stranded DNA template and a supply of nucleotides, including radioactive or labeled nucleotides to track incorporation.
4. He then analyzed the newly synthesized DNA product to detect mismatches or errors, often by using genetic assays that could reveal mutations or by biochemical methods that detect incorrect base pairing.
5. By comparing the number of correct incorporations to the number of errors, Kornberg calculated the fidelity of DNA polymerase I, often expressed as the error rate or accuracy per nucleotide incorporated.

Key Concepts

DNA Polymerase I Function

DNA Polymerase I is an enzyme responsible for synthesizing DNA by adding nucleotides complementary to a DNA template strand. It also has proofreading activity, which helps correct errors during DNA replication, ensuring accurate DNA copying.

Fidelity of DNA Replication

Fidelity refers to the accuracy with which DNA polymerases replicate DNA. High fidelity means fewer errors or mutations during DNA synthesis, which is critical for maintaining genetic stability across generations.

Experimental Assessment of Enzyme Fidelity

Kornberg assessed DNA polymerase I fidelity by measuring the frequency of errors made during DNA synthesis, often using assays that detect mismatches or mutations in the newly synthesized DNA. This involved comparing the sequence of the product to the template to quantify accuracy.