Question

How would you expect the misincorporation of bases by a DNA polymerase to change if the relative ratios of the dNTPs were \(A=T=G\) but a five-fold excess of \(C ?\)

Short answer

Answer: Altered dNTP ratios, such as the given ratio with a five-fold excess of C, can affect the accuracy of a DNA polymerase by increasing the likelihood of misincorporation events. In this case, the higher concentration of C increases the probability that the DNA polymerase may add C instead of the correct complementary base during synthesis. This leads to an increased rate of misincorporation, which negatively affects the accuracy of DNA replication.

Step-by-step solution

Role of dNTPs in DNA synthesis

The four types of dNTPs (A, T, G, C) are the building blocks for the synthesis of DNA during replication. DNA polymerase enzymes use these dNTPs to synthesize a new DNA strand complementary to the template strand, following the base-pairing rules: A pairs to T, and C pairs to G.

Misincorporation of bases

Misincorporation occurs when a DNA polymerase incorporates a non-complementary base (a mismatch) during replication. This leads to a mutation in the newly synthesized DNA strand. The accuracy of a DNA polymerase depends on its ability to distinguish correct base pairing from incorrect pairing and also on its exonuclease activity, which can excise mismatches after they have been incorporated.

Effects of altered dNTP ratios on misincorporation

If the relative ratios of the dNTPs are altered, the chances of misincorporation may increase or decrease depending on the new ratios. With the given ratio of \(A = T = G\) but a five-fold excess of \(C\), the concentration of C (cytosine) is higher relative to the other bases.

Increased misincorporation of C

Since there is a higher concentration of \(C\), DNA polymerases are more likely to incorporate C into the growing strand. This may result in more frequent misincorporation events, particularly the mispairing of C with bases other than G. The excess C increases the probability that a DNA polymerase may add C instead of the correct complementary base during synthesis.

Conclusion

In summary, the misincorporation of bases by a DNA polymerase would be expected to change if the relative ratios of the dNTPs were \(A=T=G\) but in a five-fold excess of \(C\). This is because the higher concentration of C increases the likelihood of incorporating C into the growing DNA strand, even when it is not the correct complementary base. This would result in an increased rate of misincorporation, which would affect the accuracy of DNA replication.

Key concepts

DNA Polymerase Fidelity

DNA polymerase fidelity refers to the accuracy with which a DNA polymerase enzyme synthesizes new DNA strands. This high degree of precision is crucial for maintaining the genetic integrity of organisms. DNA polymerases have a natural ability to select correct nucleotides corresponding to the template strand, ensuring that adenine (A) pairs with thymine (T), and cytosine (C) pairs with guanine (G). Nevertheless, mistakes can still occur, albeit at a low rate.

The enzyme's 'proofreading' mechanism, an inbuilt exonuclease activity, identifies and excises mistakenly incorporated nucleotides, effectively reversing the error. However, if the balance of deoxynucleoside triphosphates (dNTPs) is skewed, as would be the case with an excess of C, the enzyme might inadvertently add more C across from bases other than G, resulting in a higher misincorporation rate and potentially leading to increased mutation frequencies.

To enhance understanding, envision the fidelity of DNA polymerase as a highly meticulous editor, diligently scanning each word to ensure it's the right match, but if the editor has an oversupply of a certain letter, say 'C', they might unintentionally start slipping it into words where it doesn't belong.

dNTPs Role in Replication

Deoxynucleoside triphosphates (dNTPs) serve as the fundamental building blocks in the DNA replication process. Each dNTP consists of a nitrogenous base (A, T, C, or G), a deoxyribose sugar, and three phosphate groups. The enzyme DNA polymerase adds dNTPs to a growing DNA strand by forming a phosphodiester bond that links the sugar of one nucleotide with the phosphate group of the next one.

• A binds with T: Adenine with Thymine
• C binds with G: Cytosine with Guanine

The process is energy-driven, as the removal of two phosphates during the bonding releases energy necessary for the polymerization reaction. For DNA to be accurately replicated, it is essential that the supply of each dNTP is balanced, allowing the DNA polymerase to correctly match each base on the template strand with its complementary dNTP.

Effects of Altered dNTP Ratios

Altering the ratios of dNTPs can have substantial effects on DNA synthesis and the overall fidelity of replication. When one type of dNTP, such as cytosine in our example, is in excess, DNA polymerase may incorporate this base more often, even when it is not the correct match. This is because the relative abundance of a certain dNTP increases its likelihood of 'colliding' with the active site of the polymerase.

Consequences of dNTP Imbalance

Imbalances can lead to:

• Increased error rates in DNA replication, as the excess dNTP is more likely to be incorporated incorrectly.
• Stalling of DNA polymerase if one or more types of dNTPs are depleted, potentially leading to incomplete DNA synthesis.
• Genomic instability and mutations, which can cause various cellular dysfunctions and diseases, including cancer.

Understanding the significance of dNTP balance is akin to having the right amount of every ingredient in a recipe; too much of one can alter the flavor, or in this case, the genetic code.

Base-Pairing Rules

The base-pairing rules are fundamental to the structure and function of DNA. They describe how the nitrogenous bases pair with one another through hydrogen bonds on opposite strands of the DNA molecule. The rules follow:

• Adenine (A) pairs with Thymine (T)
• Cytosine (C) pairs with Guanine (G)

Impact of Base-Pairing Specificity

For every A in one strand, there is a T across from it in the other strand, and the same goes for pairs of C and G. This specificity ensures that the double helix structure of DNA is maintained and that during replication, the genetic information is accurately passed down. Any deviation from these rules can lead to mismatches, mutations, and possible defects in gene expression. It is as if each letter in a word has a designated partner; swapping partners would not only change the meaning of the word but could also change the entire sentence, or in the context of DNA, the genetic message.