Lecture 11 - DNA Synthesis

What is DNA metabolism?

A set of tightly regulated processes including replication, repair, and recombination that maintain and propagate genetic information.

What are the three main processes of DNA metabolism?

1) Replication 2) Repair 3) Recombination.

What are the three fundamental rules of DNA replication?

1) Replication is semiconservative. 2) Replication begins at an origin and proceeds bidirectionally. 3) DNA synthesis occurs 5'→3' and is semidiscontinuous.

Who performed the Meselson-Stahl experiment and in what year?

Matthew Meselson and Franklin Stahl, 1958.

What did the Meselson-Stahl experiment demonstrate?

That DNA replication is semiconservative.

What isotopes of nitrogen were used in the Meselson-Stahl experiment?

15N (heavy) and 14N (light).

Why would there be no intermediate hybrid band if DNA replication were conservative?

Because the old DNA would remain intact and the new DNA would be completely light, producing only two separate density bands.

What did Cairns’ experiment (1963) demonstrate?

That circular DNA replicates bidirectionally with two replication forks.

What radioactive label was used in Cairns’ experiment?

3H-thymidine (tritium-labeled thymidine).

What did Inman’s experiments show about DNA replication?

That replication begins at a unique origin in A=T rich regions, shown as 'bubbles' in denatured DNA.

What does the term 'origin of replication' mean?

A specific sequence where DNA synthesis begins.

Who discovered DNA polymerase I and when?

Arthur Kornberg, 1956.

How many DNA polymerases are present in E. coli?

At least five.

What substrates does DNA polymerase use?

Nucleoside triphosphates (dNTPs).

What provides the nucleophile in DNA elongation?

The 3’-OH group of the growing DNA strand.

What ion assists in the catalytic activity of DNA polymerase?

Mg²⁺ (magnesium ions).

What byproduct is released during DNA polymerization?

Pyrophosphate (PPi).

Why is the 3’-OH group essential for DNA synthesis?

It performs a nucleophilic attack on the α-phosphate of the incoming nucleotide.

Why does DNA polymerase require a primer?

Because it cannot initiate synthesis without a pre-existing 3’-OH group.

What can a primer be made of?

DNA or RNA (usually RNA in cells).

What are the two main regions of DNA polymerase's active site?

Insertion site (for incoming nucleotide) and postinsertion site (for newly formed base pair).

How does DNA polymerase ensure high fidelity during replication?

Its active site excludes mismatched base pairs based on geometry.

What is the approximate error rate of DNA polymerase before proofreading?

1 error per 10⁴–10⁵ base pairs.

What is the final error rate in E. coli after proofreading and repair?

1 error per 10⁹–10¹⁰ base pairs.

What enzyme activity corrects mismatched bases during replication?

3’→5’ exonuclease activity.

How does 3’→5’ exonuclease proofreading work?

It removes incorrectly paired nucleotides before DNA synthesis continues.

What additional activity does DNA polymerase I have?

5’→3’ exonuclease activity.

What is nick translation?

A process where DNA polymerase I removes nucleotides ahead of it while synthesizing new DNA, moving a strand break along the DNA.

What is the Klenow fragment?

A large fragment of DNA polymerase I containing the polymerase and 3’→5’ exonuclease activities but lacking 5’→3’ exonuclease activity.

Which enzyme proofreads DNA for mismatched base pairs?

DNA polymerase (via 3’→5’ exonuclease activity).

Which activities are present in DNA polymerase I?

5’→3’ polymerase, 3’→5’ exonuclease, and 5’→3’ exonuclease.

How does DNA polymerase prevent mismatches during synthesis?

It uses base-pair geometry recognition to exclude incorrect pairs.