In the discussion, we focused on how DNA is replicated and synthesized. We also discussed recombination at the DNA level. Along the way, we encountered many opportunities to consider how this information was acquired. On the basis of these discussions, what answers would you propose to the following fundamental questions?
How do we know that in vivo DNA synthesis occurs in the 5' to 3' direction?

Verified step by step guidance

1. Recall that DNA polymerases are the enzymes responsible for DNA synthesis during replication, and they add nucleotides to the growing DNA strand.
2. Understand that DNA polymerases require a free 3'-OH group on the primer strand to add new nucleotides, which means synthesis can only proceed by extending the strand at its 3' end.
3. Consider experimental evidence such as the use of labeled nucleotides and chain-terminating analogs (e.g., dideoxynucleotides) that show DNA synthesis proceeds by adding nucleotides to the 3' end, confirming the 5' to 3' directionality.
4. Review classic experiments like those by Arthur Kornberg and others, where the incorporation of nucleotides was measured and shown to occur only in the 5' to 3' direction, as well as the inability of DNA polymerase to add nucleotides in the 3' to 5' direction.
5. Summarize that the biochemical mechanism of DNA polymerase activity, combined with experimental labeling and termination studies, provides strong evidence that in vivo DNA synthesis occurs in the 5' to 3' direction.

Key Concepts

Directionality of DNA Synthesis

DNA synthesis occurs in the 5' to 3' direction because DNA polymerases add nucleotides to the free 3' hydroxyl group of the growing strand. This directionality is essential for the enzyme's catalytic mechanism and ensures accurate replication. Experimental evidence, such as incorporation of labeled nucleotides, supports this unidirectional synthesis.

Role of DNA Polymerase Enzymes

DNA polymerases are enzymes that catalyze the addition of nucleotides during DNA replication. They require a primer with a free 3' OH group and can only add nucleotides in the 5' to 3' direction. Their structure and function explain why DNA synthesis cannot proceed in the opposite direction.

Experimental Evidence for DNA Synthesis Direction

Classic experiments using radiolabeled nucleotides and chain-terminating analogs demonstrated that DNA synthesis proceeds 5' to 3'. For example, pulse-chase experiments and enzymatic assays showed incorporation only at the 3' end, confirming the directionality of DNA replication in vivo.