Question

Explain the effect on DNA replication of mutations that destroy each of the following activities in DNA polymerase. Also, for each kind of mutation, how might you detect the effect in an in vitro replication reaction?

a. 5′ to 3′ polymerase
b. 5′ to 3′ exonuclease
c. 3′ to 5′ exonuclease

Answer 1

Mutations that affect different DNA polymerase activities have specific consequences. Destroying the 5' to 3' polymerase activity stops DNA replication, while impairing the 5' to 3' or 3' to 5' exonuclease activity leads to the retention of RNA primers or an increase in replication errors, respectively, detectable by various molecular assays.

Step-by-step explanation:

When discussing the effects of mutations in DNA polymerase activities, it is important to address each activity separately:

5' to 3' polymerase: A mutation that destroys the enzyme's ability to add nucleotides from 5' to 3' direction would halt DNA replication altogether, as no new DNA strands could be synthesized. In an in vitro reaction, the failure to elongate the DNA strand could be detected by the absence of newly synthesized DNA.

5' to 3' exonuclease: This activity is related to the removal of RNA primers during replication. A mutation here would lead to the presence of RNA fragments in the DNA, which could be detected through sequencing or appropriate staining methods that differentiate RNA from DNA.

3' to 5' exonuclease: This is the proofreading function. A loss here increases the mutation rate because incorrect nucleotides are not excised and replaced. This effect could be detected by an increase in the error rate of the replicated DNA, observed through sequencing analysis.