Question

Blectrophilic oxidants are known to create the modified base named 7,8 -dihydro- 8 -axoguanine (oxoG) in DNA. Whereas guanine base-pairs with cytosine, oxoG base-pairs with either cytosine or adenine. (a) What are the sources of reactive oxidants within cells that cause this type of base alteration? (b) Drawing on your knowledge of nucleotide chemistry, draw the structure of $0\times 0\mathrm{G},$ and, below it, draw guanine. Opposite guanine, draw cytosine, including the hydrogen bonds that allow these two molecules to base- pair, Does the structure of oxoG, in contrast to guanine, provide any hint as to why it basepairs with adenine? (c) Assume that an unrepaired oxoG lesion is present in the helix of DNA opposite cytosine. Predict the type of mutation that will occur following several rounds of replication. (d) Which DNA repair mechanisms might work to counteract an oxoG lesion? Which of these is likely to be most effective?

Short answer

Answer: The most effective DNA repair mechanism to counteract an oxoG lesion is the base excision repair (BER) pathway, as it specifically targets and removes bases damaged by oxidation, alkylation, and deamination, which includes the oxoG lesion.

Step-by-step solution

Part (a): Sources of Reactive Oxidants in Cells

Various cellular sources of reactive oxidants include processes such as respiration in mitochondria, cytochrome P450 reactions, peroxisomes, and inflammatory responses involving NADPH oxidase and other immune cells. These oxidants can cause damage to DNA, leading to base alterations such as the formation of 7,8-dihydro-8-oxoguanine (oxoG).

Part (b): Drawing the Structures of OxoG and Guanine

First, draw the structure of 7,8-dihydro-8-oxoguanine (oxoG): 1. Begin by drawing a purine ring with the C8 position oxidized to a carbonyl group (C=O). 2. Label the atoms and add the hydrogen attached to N1 and the amino group attached to C6. 3. Draw the guanine structure below oxoG: a purine ring with a hydrogen attached to N1, an amino group attached to C6, and a keto group (C=O) attached to C6. 4. Opposite guanine, draw the structure of the cytosine: a pyrimidine ring with an amino group attached to both the C4 and N2 positions and a keto group attached to C2. 5. Add hydrogen bonds between guanine and cytosine: three dotted lines connecting guanine N1 with cytosine N3, guanine N2 with cytosine O2, and guanine O6 with cytosine N4. The structure of oxoG hints at the reason it can base pair with adenine. The C8 position in the oxoG is oxidized to a carbonyl group (C=O), which forms an additional hydrogen bond with adenine opposite to its N7 position. This allows for oxoG to potentially base pair with both cytosine and adenine.

Part (c): Predicting the Type of Mutation with an Unrepaired OxoG Lesion

If an unrepaired oxoG lesion is present in the DNA helix opposite to cytosine and several rounds of replication occur, it could lead to a transversion mutation. This is because oxoG can base pair with either cytosine or adenine. If oxoG is paired with adenine during replication, this will lead to a G:C to T:A transversion mutation.

Part (d): DNA Repair Mechanisms to Counteract OxoG Lesion

Two main DNA repair mechanisms can work to counteract an oxoG lesion: base excision repair (BER) and nucleotide excision repair (NER). 1. Base excision repair (BER) involves the activity of DNA glycosylase, which recognizes and removes the damaged base, followed by endonuclease cleavage, DNA polymerase filling in the gap, and final ligation by DNA ligase. 2. Nucleotide excision repair (NER) recognizes and removes a broad range of damaged bases by cleaving on both sides of the damage, removing a single-stranded DNA segment, and then using DNA polymerase and ligase to fill the gap. The base excision repair (BER) mechanism is more likely to be effective in counteracting an oxoG lesion because it is a more specific repair pathway that targets and removes bases damaged by oxidation, alkylation, and deamination, such as the oxoG lesion.

Key concepts

7,8-Dihydro-8-Oxoguanine (oxoG)

7,8-dihydro-8-oxoguanine, commonly known as oxoG, is a pernicious DNA lesion resulting from the oxidative damage to guanine, one of the four main bases in DNA. Oxygen free radicals generated from various cellular processes, like mitochondrial respiration or inflammatory responses, can lead to the formation of oxoG. This oxidized derivative of guanine is particularly concerning because it has an unusual ability to base-pair with adenine in addition to cytosine, its normal partner, hence triggering mutations.

Imagine the typical guanine-cytosine pairing as a neatly matched couple. When guanine is replaced by oxoG, it's as if a third party, akin to adenine, could also pair up, undermining the fidelity of DNA replication. Over time, this mispairing capacity of oxoG may cause transversion mutations, where a purine base is replaced by a pyrimidine base, or vice-versa, which can lead to serious genetic consequences if not repaired.

Base Excision Repair (BER)

Base Excision Repair (BER) is akin to a precise surgical strike where a single damaged base in the DNA is targeted and removed. The process involves specialized enzymes called DNA glycosylases which scan the DNA and recognize abnormal bases like oxoG. Once identified, the glycosylase snips off the damaged base, creating an abasic site (a site without a base).

Subsequently, the BER pathway calls upon additional players; an endonuclease cuts the DNA backbone at the abasic site, a DNA polymerase then inserts a correct base, and DNA ligase seals the deal by repairing the nick in the backbone. Together, these orchestrated steps correct the error without much ado, maintaining genomic integrity. BER is usually the best at keeping cellular DNA clean of oxoG, demonstrating its crucial role in DNA caretaking.

Nucleotide Excision Repair (NER)

Nucleotide Excision Repair (NER) is a more encompassing DNA repair mechanism when compared to BER. This versatile repair process is engineered to recognize a wide array of DNA lesions that cause bulky distortions in the DNA helix, such as those caused by UV light damage.

The NER process demarcates its target by detecting distorted DNA, then dispatching a team of proteins to excise a segment of the single-stranded DNA encompassing the lesion. This gap is swiftly filled in by leveraging the template strand and the concerted action of DNA polymerase and DNA ligase. While NER is a broad-spectrum DNA repair pathway, it is not the primary mechanism to resolve smaller, less-distorting lesions such as oxoG, thus highlighting the extensive toolbox our cells possess to protect our genetic material.

Transversion Mutation

A transversion mutation is a special kind of genetic malfunction where a purine base (adenine or guanine) is swapped with a pyrimidine base (cytosine or thymine) or vice versa. This diagonal swap in the DNA helix can lead to drastic changes because it replaces a base with a structurally dissimilar one.

When oxoG pairs with adenine during replication instead of cytosine, the stage is set for a transversion. The subsequent DNA replications can cement this error, leading from a G:C pair to a T:A pair. This G to T transversion mutation is a classic example of a permanent change that can have profound effects, ranging from disrupted protein function to the activation of oncogenes. Understanding transversion mutations is pivotal since they account for many of the mutations related to cancer and genetic disorders.