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 dnaE 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

Answer: A mutation in the dnaE gene affects the α subunit of DNA polymerase III, leading to impaired or completely inactive DNA synthesis during replication. The mutant strain can be maintained by introducing a plasmid containing a functional copy of the dnaE gene. A mutation in the dnaQ gene affects the ε subunit of DNA polymerase, causing reduced proofreading capability during DNA replication. The mutant strain can be maintained by introducing a plasmid containing a functional copy of the dnaQ gene.

Step-by-step solution

(a) Effect of a mutation in the dnaE gene

A mutation in the dnaE gene would affect the α subunit of DNA polymerase III, which has the crucial function of adding nucleotides to the growing DNA chain during replication. A mutation in this gene could lead to a defective α subunit, resulting in impaired or completely inactive DNA polymerase III. This would mean that the E. coli strain would have difficulty or be unable to synthesize DNA during replication, leading to severe growth defects or even cell death.

(a) Maintaining the mutant strain

To maintain a mutant strain with an impaired α subunit of DNA polymerase III, the strain must be supplemented with a functional copy of the dnaE gene. This can be achieved by introducing a plasmid containing a functional dnaE gene into the defective strain. By providing the mutant strain with the necessary dnaE gene product, it would be able to synthesize DNA and grow under normal conditions.

(b) Effect of a mutation in the dnaQ gene

The dnaQ gene encodes the ε subunit of DNA polymerase, which is responsible for providing the 3'-5' exonuclease activity of the enzyme. This activity is crucial for proofreading during replication, as it allows for the removal of incorrect nucleotides that have been added to the growing DNA chain. A mutation in the dnaQ gene can result in a defective ε subunit, leading to a reduced or lost proofreading capability of the DNA polymerase. The consequence would be reduced fidelity during DNA replication and an increased frequency of mutations in the synthesizing DNA.

(b) Maintaining the mutant strain

A mutant strain with a defective ε subunit of DNA polymerase can be maintained similarly by introducing a plasmid containing a functional copy of the dnaQ gene into the strain. The functional ε subunit produced by the foreign dnaQ gene would complement the defective gene product in the mutant strain, thereby restoring the proofreading activity and allowing for accurate DNA replication and normal growth.

Key concepts

DNA polymerase III

DNA polymerase III is a vital enzyme responsible for DNA replication. It's like a construction crew, methodically building the DNA molecule by adding nucleotides to the growing chain. This enzyme is composed of multiple subunits, each performing a specific function. Among these, the α (alpha) subunit acts as the main builder, while the ε (epsilon) subunit performs quality checks by proofreading the newly added nucleotides for errors. Being the primary enzyme complex in bacterial DNA replication, DNA polymerase III plays a critical role in ensuring that cells divide accurately. Any malfunction or mutation affecting its function, especially in these subunits, can lead to faulty DNA replication, which might have severe consequences for the cell's health and ability to reproduce. It's crucial for maintaining the genetic integrity of an organism, which is why its accurate function is so important, especially in microorganisms like E. coli that rely on rapid and accurate cell division.

E. coli genetics

E. coli, a common bacterium found in the gut, is a key player in genetic research. Its simple genetic makeup and rapid replication cycle make it an ideal model for studying fundamental biological processes, including DNA replication. Researchers have been able to identify many genes involved in DNA synthesis by observing E. coli strains with mutations. In these studies, scientists isolate mutant strains that struggle or fail to synthesize DNA under specific conditions. These strains serve as a window into understanding how genes and their products, such as those related to DNA polymerase III, contribute to DNA replication. The relatively small genome of E. coli allows researchers to manipulate its genes easily and observe the effects of specific mutations, enhancing our understanding of bacterial genetics and gene function. These experiments provide insight not only into bacterial genetics but also into broader biological functions given the conserved nature of many genes across different organisms.

Gene mutation effects

Gene mutations can have broad and varied effects, depending on the gene affected and the role it plays within the cell. When it comes to DNA replication, mutations can severely impact the process. A mutation in genes like dnaE or dnaQ, both of which code for subunits of DNA polymerase III, can disrupt the enzyme's function. Mutations can lead to defective proteins unable to perform their intended duties. For instance, a defective α subunit from a dnaE gene mutation might halt DNA synthesis, while a defective ε subunit from a dnaQ mutation could impede error-checking, leading to more mutations. Understanding the outcomes of these mutations helps in unraveling biological processes like error correction during DNA replication and illustrates the genetic checks that prevent genomic instability, which is a precursor for many diseases and conditions.

dnaE gene

The dnaE gene codes for the α subunit of DNA polymerase III, the main component responsible for synthesizing new DNA strands. This subunit is akin to the worker bee in the DNA replication process, tirelessly adding nucleotides to the growing chain. A mutation in the dnaE gene can compromise the functionality of this α subunit. If it's faulty or inactive, the polymerase struggles to add nucleotides properly, leading to severe replication issues. As a result, the E. coli cells may experience inhibited growth or even cell death if the mutation isn't compensated for. To maintain a mutant strain with a dnaE gene defect, scientists often introduce a plasmid carrying a functional dnaE gene. This extra piece of DNA compensates for the mutant gene, ensuring the α subunit is present and functional so that DNA replication can proceed smoothly.

dnaQ gene

The dnaQ gene encodes the ε subunit of DNA polymerase III, which acts as a proofreader during DNA replication. Its role is to correct mistakes by removing incorrectly paired nucleotides, thereby preventing mutations. This proofreading ability is crucial for the high fidelity of DNA replication. A mutation in the dnaQ gene, however, may render the ε subunit ineffective. Without its error-correcting function, DNA polymerase III may incorporate incorrect nucleotides, leading to increased mutational errors in the DNA. Over time, this can affect the stability of the organism's genome, potentially causing malfunction in essential cellular processes or even cell death. To address this, scientists can introduce a plasmid with a functional dnaQ gene into the mutant strain. This genetic supplementation compensates for the defective gene and restores proofreading, thus enabling err-free replication and the maintenance of genomic integrity.