Question

Genetics Questions

1) What does “proofreading” refer to with regard to DNA replication? Briefly explain its mechanism.

2) All known DNA polymerases catalyze synthesis only in the 5' ? 3' direction. Nevertheless, during semiconservative DNA replication in the cell, they are able to catalyze the synthesis of
both daughter chains, which would appear to require synthesis in the 3' ? 5' direction. Explain the process that occurs in the cell that allows for synthesis of both daughter chains by DNA
polymerase.

Answer 1

1) Proofreading

Proofreading, as the name suggests, regers to the process of correcting any error introduced during the process of replication. DNA synthesizing enzymes, also known as DNA polymerases possess the ability to check the bases introduced during replication and remobe/replace the incorrect nucleotide before further synthesis. This removal is due to the presence of 3' to 5' exponuclease activity and is necessary for the high degree of fidelity characteristic of DNA replication.

When presented with a template-primer DNA having a terminal mismatch, the 3' to 5' exonulcease activity of the polymerase chops off the unpaired base. Introduction of a mismatched base will inhibit the catalyzation of covalent extension. Followed by the removal of mismatched nucleotide, the strand will be presented with a correct base paired primer terminus and DNA polymerase will catalyze 5' to 3' covalent extension.

2) All DNA polymerases have an absolute requirement for a 3'-hydroxyl end and carry out 5' to 3' synthesis. These polymerases only catalyze covalent extension in the 5' to 3' direction. Thus, the synthesis of the strand growing in the overall 3' to 5' direction is discontinuous. Short segments are synthesized in the 5' to 3' direction and then covalently joined by polynucleotide ligase.

Since the complementary strands of DNA have opposite polarity, one strand is extended in overall 5' to 3' direction while the other in 3' to 5' direction. A short RNA strand is synthesized to provide a 3'-OH primer for DNA synthesis. This RNA primer is subsequently removed and replaced with DNA by the 5' to 3' exonuclease and 5' to 3' polymerase activity built into DNA polymerase I. DNA ligase then covalently closes the nascent DNA chain, catalyzing the formation of phosphodiester linkages between adjacent 3'-hydroxyls and 5'-phosphates.