Question

You are a scientist studying DNA replication. You have the ability to introduce temperature-sensitive mutations into the bacteria that you are studying (temperature-sensitive mutations work normally at one temperature, but when you raise the temperature a little, the mutation exerts its effect. This allows you to grow colonies with mutations that would otherwise be lethal. The fact that these mutations are temperature sensitive has nothing to do with the problem, other than to make it accurate to how these would actually be done. In other words, don't worry about temperature-sensitivity at all. Just think about possible mutations).

You want to mutate bacteria so that they will have the following phenotypes:

1. Very slow DNA replication
2. DNA replication with a lower mutation rate
3. A mutation where when the cell replicates its DNA, it dies.

Please come up with two different targets of mutation for each phenotype. Explain how each mutation would work, why you chose them, and any side effects there would be.

Answer 1

Two mutation targets for achieving very slow DNA replication include the mutations in genes PCLAF and E2F1 encoding PCNA (Proliferating Cell Nuclear Antigen) and the transcription factor E2F1 respectively. PCNA, a clamp like molecule serves as a scaffold for the DNA polymerases involved in DNA replication during the S phase and E2F is responsible for the transcription of PCNA.

The targets for lowering the mutation rate during DNA replication are DNA polymerases class A and B. Since they are invloved in the fidelity (proof-reading) of DNA replication process. DNA polymerase Class A function as DNA repair proteins and DNA polymerase class B function primarily in genomic replication.

The targets for mutation for the cell death while it replicates include the Caspases and Bcl-2 family proteins. Caspases function as processors of pro-inflammatory cytokines that initiate cell apoptosis (death). Bcl-2 family of proteins consisting of pro-apoptotic members Bak and Bax proteins which induce the cytochrome c release from mitochondria disrupting the Electron Transport Chain leading to cell apoptosis.