Question

Many of the gene products involved in DNA synthesis were initially defined by studying mutant \(E .\) coli strains that could not synthesize DNA. (a) The \(d n a E\) gene encodes the \(\alpha\) subunit of DNA polymerase III. What effect is expected from a mutation in this gene? How could the mutant strain be maintained? (b) The \(d n a Q\) gene encodes the \(\varepsilon\) subunit of DNA polymerase. What effect is expected from a mutation in this gene?

Short answer

Short Answer: Mutations in the dnaE and dnaQ genes in E. coli impair the DNA polymerase III's polymerization and proofreading activities, respectively. For the dnaE mutant strain, maintenance can involve providing a rich medium that supports growth and using a suppressor mutation or an extrachromosomal copy of the wild-type dnaE gene. For the dnaQ mutant strain, maintenance can involve genetic complementation with a functional dnaQ gene or another gene encoding a protein with similar proofreading activity, or maintaining under conditions that minimize replication errors and promote DNA repair.

Step-by-step solution

The α subunit of DNA polymerase III, encoded by the dnaE gene, is responsible for the polymerization activity of the enzyme. It adds nucleotides to the growing DNA strand during DNA replication, ensuring the accurate copying of genetic information. #Step 2: Effect of a mutation in the dnaE gene#

A mutation in the dnaE gene, which encodes the α subunit of DNA polymerase III, would impair or abolish the polymerization activity of the enzyme. This means that DNA replication would be compromised or completely halted, affecting the cell's ability to reproduce and potentially leading to cell death. #Step 3: Maintaining the mutant strain with a mutation in the dnaE gene#

In order to maintain a mutant strain with a mutation in the dnaE gene, conditions must be provided that would allow the cells to survive and grow despite their inability to synthesize DNA properly. One possibility is to maintain the cells in a rich medium that provides all the necessary nutrients for growth, and by using a suppressor mutation that restores partial or full function to the α subunit. Another possibility is to provide an extrachromosomal copy of the wild-type dnaE gene to the cells, allowing them to express a functional α subunit and replicate their DNA. #Step 4: Understanding the role of the ε subunit of DNA polymerase III#

The ε subunit of DNA polymerase III, encoded by the dnaQ gene, is responsible for the proofreading activity of the enzyme. It removes any mismatched nucleotides incorporated during DNA replication, ensuring the fidelity of the process and preventing the accumulation of errors in the genetic code. #Step 5: Effect of a mutation in the dnaQ gene#

A mutation in the dnaQ gene, which encodes the ε subunit of DNA polymerase III, would impair or abolish the proofreading activity of the enzyme. This means that the accuracy of DNA replication would be reduced, leading to a higher rate of errors (mutations) in the DNA sequence. Such mutations could potentially be detrimental to the cell and its offspring. #Step 6: Maintaining the mutant strain with a mutation in the dnaQ gene#

To maintain a mutant strain with a mutation in the dnaQ gene, one approach could be to provide an external source of the wild-type ε subunit, or a functional equivalent, to compensate for the defective proofreading activity. This could be achieved through genetic complementation, for example by transforming the cells with a plasmid containing a functional dnaQ gene or another gene encoding a protein with similar proofreading activity. Alternatively, the cells could be maintained under conditions that minimize the occurrence of replication errors and/or promote the repair of such errors.

Key concepts

DNAE Gene Function

At the heart of DNA replication lies the DNA polymerase III, a critical enzyme complex responsible for copying a cell’s genetic blueprint accurately. The dnaE gene plays a pivotal role as it encodes the alpha subunit, which is the chief architect of this process. The alpha subunit is tasked with adding nucleotides, the building blocks of DNA, to form a new strand by following the template provided by the original strand.

Understanding its function is fundamental – any mutation in the dnaE gene could severely impact the replication process. Such mutations might result in the inability to synthesize DNA correctly, posing a threat to cell viability and proliferation. Consequently, mutations in dnaE could lead to severe growth defects or cell death.

DNAQ Gene Function

Ensuring that the genetic information is replicated without errors is a monumental task, given the vast number of nucleotides to be copied. This is where the epsilon subunit, encoded by the dnaQ gene, plays its part. It acts as a guardian of genome integrity by proofreading the new DNA strands. As the alpha subunit adds nucleotides, the epsilon subunit scans for mismatches and excises incorrectly paired bases.

A mutation in the dnaQ gene, therefore, has the potential to compromise DNA replication fidelity. It could impair the enzyme's ability to detect and correct errors, leading to an increased mutation rate. This could ultimately affect the organism’s survival and adaptation, as the accumulation of genetic errors can have far-reaching consequences.

DNA Replication Fidelity

DNA replication fidelity is the accuracy with which DNA is copied during cell division. A high degree of fidelity is crucial for the maintenance of genetic stability across generations. The intricate machinery of DNA polymerase III harnesses both the alpha and epsilon subunits to ensure that copying errors are minimized.

The importance of this fidelity cannot be understated, as even slight errors can result in mutations that might lead to diseases, such as cancer, or other genetic disorders. The proofreading activity of the epsilon subunit of DNA polymerase III, in conjunction with other repair mechanisms, helps to maintain a remarkably low error rate during DNA replication.

Mutant Strain Maintenance

When mutations occur in critical genes like dnaE or dnaQ, researchers may want to keep the mutant strains alive for study. Maintaining such strains often involves creative biological techniques. For instance, a rich medium can provide essential nutrients that support growth despite compromised DNA replication. Alternatively, suppressor mutations might rescue the functionality of the mutated protein, or an extrachromosomal element bearing the wild-type gene can be introduced to the cell to compensate for the defective gene.

Where the dnaQ gene is concerned, a similar strategy of genetic complementation can be used. Introducing a plasmid that provides a functional proofreading subunit can sustain the cell despite the native subunit being non-functional. These approaches allow scientists to continue studying mutational effects and their mechanisms, which is essential for understanding the robustness of DNA replication and the implications of its failure.