Question

Several temperature-sensitive mutant strains of \(E .\) coli display the following characteristics. Predict what enzyme or function is being affected by each mutation. (a) Newly synthesized DNA contains many mismatched base pairs. (b) Okazaki fragments accumulate, and DNA synthesis is never completed. (c) No initiation occurs. (d) Synthesis is very slow. (e) Supercoiled strands remain after replication, which is never completed.

Short answer

Question: Based on the given characteristics, predict the enzyme or function affected by each mutation in temperature-sensitive mutant strains of E. coli. a) Presence of many mismatched base pairs in newly synthesized DNA. b) Accumulation of Okazaki fragments and incomplete DNA synthesis. c) Absence of initiation. d) Slow synthesis of DNA. e) Persistence of supercoiled strands after replication and incomplete replication.

Step-by-step solution

Mutation (a)

The characteristic mentioned for this mutation is the presence of many mismatched base pairs in newly synthesized DNA. This indicates that there might be a problem with the enzyme responsible for proofreading and repairing errors in DNA synthesis. The enzyme responsible for this function is DNA polymerase's exonuclease domain. Prediction: The mutation affects the exonuclease domain of DNA polymerase.

Mutation (b)

The characteristic mentioned for this mutation is the accumulation of Okazaki fragments and incomplete DNA synthesis. Okazaki fragments are synthesized on the lagging strand during DNA replication and later joined by an enzyme called DNA ligase. If there is a problem with this enzyme, Okazaki fragments won't be joined, and DNA synthesis cannot be completed. Prediction: The mutation affects DNA ligase.

Mutation (c)

The characteristic mentioned for this mutation is the absence of initiation. The initiation of DNA replication requires an enzyme called DNA helicase, along with other proteins (like DnaA and DnaC), to open up the DNA double helix and start the replication process. If there is a problem with any of these proteins, initiation won't occur. Prediction: The mutation affects DNA helicase, DnaA, or DnaC.

Mutation (d)

The characteristic mentioned for this mutation is the slow synthesis of DNA. One possibility is that there is a problem with DNA polymerase responsible for the elongation of the leading and lagging strands. Another possibility could be a mutation affecting the processivity of the enzyme, which depends on the sliding clamp, a protein complex that keeps the DNA polymerase on the DNA strand during replication. Prediction: The mutation affects DNA polymerase and/or the sliding clamp.

Mutation (e)

The characteristic mentioned for this mutation is the persistence of supercoiled strands after replication and incomplete replication. Supercoiled DNA is uncoiled by an enzyme called DNA topoisomerase, which relaxes the supercoils by breaking and rejoining the DNA strands. If there is a problem with this enzyme, supercoils will not be resolved, and replication cannot be completed. Prediction: The mutation affects DNA topoisomerase.

Key concepts

DNA Polymerase Exonuclease Activity

DNA replication is a highly accurate process, crucial for the transfer of genetic information from one generation to another. A key player in this procedure is DNA polymerase, which not only synthesizes new DNA strands but also ensures fidelity through its exonuclease activity. This proofreading function involves removing nucleotides that have been incorrectly inserted.

Analogous to an editor scrutinizing a text for typos, exonuclease activity corrects errors by excising mismatched base pairs. During DNA synthesis, the enzyme adds nucleotides to the growing strand. If an incorrect nucleotide is added, DNA polymerase senses this because of improper base pairing. The polymerase then uses its 3' to 5' exonuclease activity to remove the mismatched nucleotide. After this, synthesis resumes with the correct nucleotide being incorporated.

Proofreading and Mutation Consequences

Without proper exonuclease activity, the error rate during DNA replication would increase. This can lead to mutations, which are permanent alterations in the DNA sequence. If such mutations occur within genes, they may result in malfunctioning proteins, which can cause diseases or developmental issues in organisms. The exercise provided highlights this by linking a high frequency of mismatched base pairs to a potential mutation affecting the exonuclease domain of DNA polymerase.

DNA Ligase and Okazaki Fragments

In DNA replication, the lagging strand is synthesized in short segments known as Okazaki fragments. These fragments must be connected to form a continuous DNA strand, a critical step performed by the enzyme DNA ligase.

DNA ligase acts like a molecular gluestick, sealing 'nicks' or gaps in the DNA. It catalyzes the formation of a phosphodiester bond between the adjacent nucleotides, efficiently linking Okazaki fragments on the lagging strand. This enzyme's role ensures that DNA's double helix structure remains intact after replication.

Implications of Ligase Malfunction

Without a functioning DNA ligase, the continuity of the DNA strand would be compromised, resulting in an accumulation of Okazaki fragments. This condition would halt the replication process, as seen in the exercise where the presence of numerous unjoined Okazaki fragments suggests a possible mutation in DNA ligase.

DNA Helicase Initiation

Initiation of DNA replication is a complex process that begins with the unwinding of the double helical structure of DNA. DNA helicase is pivotal in this step, as it separates the two strands of DNA, creating a replication fork where DNA polymerases can start synthesizing new strands.

DNA helicase is essentially the zipper of the replication process; it travels along the DNA, breaking hydrogen bonds between the bases and opening the double helix. This action requires energy, which the helicase obtains by hydrolyzing ATP.

Hindrances to Initiation and Cellular Consequences

When DNA helicase is not functioning correctly—due to, for example, a mutation—initiation cannot occur, resulting in halted replication. This would lead to cellular dysfunction as DNA replication is a prerequisite for cell division. The inability to initiate replication, as suggested in the exercise by the lack of initiation, points towards a malfunction in DNA helicase or associated proteins required for the commencement of replication.

DNA Topoisomerase Supercoiling

DNA topoisomerase is an essential enzyme that solves the problem of DNA supercoiling, a stress introduced to DNA's structure during replication and transcription. As the helix unwinds, supercoiling can occur ahead of the replication fork, which can hinder the progress of the replication machinery.

DNA topoisomerase prevents supercoils from becoming too tight by cutting the DNA strands and then rejoining them after allowing for the necessary unwinding. This process is akin to relieving tension on a twisted telephone cord by permitting it to spin.

The Detriments of Topoisomerase Deficiency

Deficiencies in topoisomerase function, as mentioned in the exercise, lead to the persistence of supercoiled DNA which can stall replication. Such a scenario underscores how mutations in the topoisomerase genes could result in the accumulation of supercoils and incomplete DNA replication, potentially causing widespread genome instability.