Question

You have generated a mutant strain of eukaryotic cells that constitutively express proteins required for translesion DNA synthesis (TLS). Would these cells have a mutator phenotype? Explain. One of the strains that you are working with shows an additional mutation whereby the processivity of a TLS polymerase is increased. What would be the consequence of this mutation?

Short answer

Answer: Yes, eukaryotic cells that constitutively express proteins required for TLS exhibit a mutator phenotype due to the error-prone nature of TLS polymerases. A mutation that increases the processivity of a TLS polymerase would further exacerbate the mutator phenotype, leading to an even higher mutation rate in these cells.

Step-by-step solution

Understanding translesion DNA synthesis (TLS) and mutator phenotype

Translesion DNA synthesis (TLS) is a DNA damage tolerance mechanism where specialized DNA polymerases carry out replication across damaged DNA sites, even at the expense of fidelity. DNA polymerases involved in TLS are typically error-prone, meaning that they tend to incorporate incorrect nucleotides, thus increasing the mutation rate. A mutator phenotype is a term used to describe cells that show an increase in the mutation rate.

Determining if cells with constitutive TLS expression have a mutator phenotype

Since TLS polymerases are error-prone, the constitutive expression of proteins required for TLS would lead to increased chances of incorporating incorrect nucleotides during DNA replication. This would increase the mutation rate in eukaryotic cells. As a result, these mutant TLS-expressing cells would indeed have a mutator phenotype.

Explaining the effect of increasing TLS polymerase processivity

Processivity refers to the ability of a polymerase enzyme to hold onto and continue synthesizing DNA without disassociating from the template. An increased processivity of a TLS polymerase means that it will replicate DNA more efficiently across damaged sites without falling off the DNA strand. However, given the error-prone nature of the TLS polymerases, this would lead to an even higher rate of incorrect nucleotide incorporation and, consequently, more mutations. The consequence of this mutation would be a further exacerbation of the mutator phenotype, increasing the mutation rate even more in these cells.

Key concepts

Mutator Phenotype

When a eukaryotic cell exhibits a high frequency of mutations, it is said to have a mutator phenotype. Mutations are changes in the DNA sequence that can lead to genetic diversity; however, when they occur too often, they might result in genetic instabilities and various diseases, including cancer. This phenotype can occur naturally, or it may be induced by environmental factors such as radiation or chemicals.

In the context of translesion DNA synthesis (TLS), the mutator phenotype becomes particularly relevant. TLS allows cells to replicate DNA despite the presence of DNA lesions, by using specialized error-prone DNA polymerases. While these polymerases help the cell to overcome the obstacle of damaged DNA, they lack the proofreading abilities of regular polymerases and introduce mutations more frequently during DNA replication. Thus, constitutive expression of TLS proteins can lead to an elevation in the baseline mutation rate, characteristic of a mutator phenotype. Not all mutations are deleterious; some may confer advantages, such as drug resistance in microbes or adaptation in a changing environment. Nevertheless, the overall increase in mutation rate can be harmful to the organism's genetic integrity.

DNA Polymerase Processivity

The concept of DNA polymerase processivity refers to the ability of a DNA polymerase to catalyze the addition of nucleotides without disassociating from the DNA template. Processivity is a critical factor for the efficiency of DNA replication. In simple terms, a highly processive polymerase can add many nucleotides to a growing DNA strand before letting go, much like a train traveling a long distance without stopping.

In the case of TLS polymerases, they normally exhibit lower processivity compared to the regular DNA polymerases that conduct the bulk of DNA replication. This can be seen as a safeguard, limiting the extent of errors introduced into the genome. However, when a mutation increases the processivity of a TLS polymerase, it grants the enzyme an enhanced ability to synthesize longer stretches of DNA without detaching. This change could theoretically make DNA replication more robust in the face of damage, but it also allows for more errors to be introduced before the enzyme is 'switched out' for a more accurate polymerase. The impact of this mutation ultimately depends on a delicate balance—while it may confer an ability to bypass more extensive DNA damage, it also risks further amplifying the mutator phenotype, potentially jeopardizing genomic stability.

DNA Damage Tolerance

The cellular capability to cope with and survive DNA damage, without immediately correcting the underlying damage, is known as DNA damage tolerance. There are multiple pathways by which cells achieve this, and one such mechanism is translesion DNA synthesis (TLS). TLS is a stopgap measure, allowing cells to duplicate their genome and continue to divide even in the presence of DNA lesions.

DNA damage tolerance is essential for life, as DNA can be damaged by a variety of internal and external factors, including metabolic by-products, UV light, and various chemicals. Without mechanisms to tolerate damage, cells could either stall indefinitely, leading to cell death or senescence, or initiate error-prone repair processes that could culminate in severe genetic abnormalities.

Translesion DNA synthesis is a double-edged sword. On one side, it prevents the stalling of the replication fork, which could be lethal to the cell. On the other, it does so by allowing specialized polymerases, which are inherently less accurate, to add nucleotides opposite damaged bases. This process, while promoting cell survival, can lead to permanent changes in the DNA sequence if the damage is not repaired later on, potentially contributing to a mutator phenotype. It illustrates the balance organisms must maintain between preserving genetic stability and allowing for enough flexibility to manage the diverse insults that their DNA can face.