Enzymes That Cut Out Damaged Sections Of Dna

DNA, the blueprint of life, is constantly under attack. From environmental toxins to the very processes of cellular metabolism, numerous factors can inflict damage on this crucial molecule. Fortunately, our cells possess remarkable repair mechanisms, including specialized enzymes that act like molecular surgeons, excising damaged sections of DNA to maintain the integrity of our genetic code. These enzymes are critical for preventing mutations, which can lead to diseases like cancer and premature aging.

Understanding DNA Damage and Repair

DNA damage is a broad term encompassing any chemical alteration to the DNA molecule. This damage can manifest in various forms, including:

• Base modifications: Changes to the chemical structure of individual DNA bases (adenine, guanine, cytosine, and thymine).
• Strand breaks: Discontinuities in the sugar-phosphate backbone of the DNA molecule. These can be single-strand breaks (SSBs) or double-strand breaks (DSBs).
• Crosslinks: Abnormal covalent bonds between DNA strands or between DNA and proteins.
• Bulky adducts: Large chemical groups attached to DNA bases, distorting the DNA structure.

These damages arise from a multitude of sources, both internal and external:

• Reactive oxygen species (ROS): Byproducts of normal cellular metabolism that can damage DNA bases.
• Ultraviolet (UV) radiation: Exposure to sunlight can cause the formation of pyrimidine dimers, which distort the DNA helix.
• Ionizing radiation: X-rays and gamma rays can induce strand breaks and base modifications.
• Chemical mutagens: Substances like бензопирен (found in cigarette smoke) and alkylating agents can react with DNA, causing mutations.
• Replication errors: Mistakes made during DNA replication can lead to mismatched bases or insertions/deletions.

To combat this constant barrage of damage, cells have evolved sophisticated DNA repair pathways. One of the most important of these pathways involves enzymes that specifically recognize and remove damaged sections of DNA.

Key Enzymes Involved in DNA Excision Repair

Several families of enzymes play crucial roles in excising damaged sections of DNA. These include:

• DNA glycosylases: These enzymes initiate base excision repair (BER) by recognizing and removing damaged or modified bases from the DNA backbone.
• Endonucleases: These enzymes cleave the phosphodiester bond within a DNA strand, allowing for the removal of damaged or mismatched sequences.
• Exonucleases: These enzymes remove nucleotides from the ends of DNA strands, working in either a 5' to 3' or 3' to 5' direction.
• Flap endonucleases (FEN1): These enzymes specifically cleave flap structures that arise during long-patch base excision repair and Okazaki fragment processing during DNA replication.

Let's explore these enzymes and their roles in greater detail.

1. DNA Glycosylases: The Base Excision Repair Initiators

DNA glycosylases are a family of enzymes responsible for initiating the base excision repair (BER) pathway, a critical mechanism for removing damaged or modified bases from DNA. These enzymes possess a remarkable ability to scan the DNA double helix, identify aberrant bases, and selectively cleave the N-glycosidic bond that connects the base to the sugar-phosphate backbone.

How DNA Glycosylases Work:

1. Damage Recognition: DNA glycosylases employ a variety of mechanisms to recognize specific types of base damage. Some glycosylases have a broad substrate specificity, while others are highly specific for a particular type of lesion. The recognition process often involves the enzyme flipping the damaged base out of the DNA helix, allowing it to fit into the enzyme's active site.
2. Base Removal: Once the damaged base is recognized and bound, the glycosylase catalyzes the hydrolysis of the N-glycosidic bond, releasing the damaged base from the DNA. This creates an apurinic/apyrimidinic (AP) site, also known as an abasic site, which is a sugar-phosphate residue lacking a base.
3. AP Site Processing: The AP site is then processed by other enzymes in the BER pathway, including AP endonucleases, which cleave the DNA backbone at the AP site, and DNA polymerases, which fill the gap with the correct nucleotide.

Examples of DNA Glycosylases and Their Specificities:

• UNG (Uracil-DNA Glycosylase): Removes uracil from DNA. Uracil can arise in DNA through the deamination of cytosine or the incorporation of dUTP during DNA replication. UNG is crucial for preventing mutations caused by uracil mispairing with adenine.
• OGG1 (8-Oxoguanine DNA Glycosylase): Removes 8-oxoguanine (8-oxoG), a common form of oxidative DNA damage caused by reactive oxygen species. 8-oxoG can mispair with adenine, leading to transversion mutations.
• MYH (MutY Adenine Glycosylase): Removes adenine that is mispaired with 8-oxoG. MYH works in conjunction with OGG1 to prevent mutations caused by 8-oxoG.
• TDG (Thymine DNA Glycosylase): Removes thymine that is mispaired with guanine. This mismatch can arise from the deamination of 5-methylcytosine.

The Importance of BER:

The base excision repair pathway initiated by DNA glycosylases is essential for maintaining genomic stability. Defects in BER can lead to an accumulation of DNA damage, increased mutation rates, and an elevated risk of cancer and other diseases. For example, mutations in the MUTYH gene, which encodes MYH, are associated with an increased risk of colorectal cancer.

2. Endonucleases: Cleaving Within the DNA Strand

Endonucleases are enzymes that cleave the phosphodiester bond within a DNA strand, as opposed to exonucleases, which remove nucleotides from the ends of DNA strands. Endonucleases play crucial roles in various DNA repair pathways, including nucleotide excision repair (NER), mismatch repair (MMR), and double-strand break repair (DSBR).

Types of Endonucleases and Their Functions:

• AP Endonucleases: These enzymes, such as APE1 (apurinic/apyrimidinic endonuclease 1), cleave the DNA backbone at AP sites created by DNA glycosylases during BER. APE1 is the major AP endonuclease in human cells and is essential for processing AP sites and initiating the subsequent steps of BER.

• Structure-Specific Endonucleases: These enzymes recognize and cleave specific DNA structures, such as:

  • XPF-ERCC1: A complex involved in nucleotide excision repair (NER). It cleaves the damaged DNA strand at the 5' side of the lesion.
  • XPG: Another NER endonuclease that cleaves the damaged DNA strand at the 3' side of the lesion.
  • MUS81-EME1: A structure-specific endonuclease involved in resolving stalled replication forks and processing DNA intermediates during homologous recombination.

• Restriction Endonucleases: While primarily known for their role in bacterial defense against viral DNA, restriction endonucleases are also used extensively in molecular biology for cutting DNA at specific sequences. They are not directly involved in DNA repair but are valuable tools for manipulating DNA in research settings.

Endonucleases in Nucleotide Excision Repair (NER):

NER is a major DNA repair pathway that removes bulky DNA lesions, such as pyrimidine dimers caused by UV radiation and chemical adducts. The NER pathway involves the following steps:

1. Damage Recognition: NER proteins recognize and bind to the damaged DNA site.
2. Dual Incision: The endonucleases XPF-ERCC1 and XPG make incisions on either side of the lesion, creating a short DNA fragment containing the damage.
3. Fragment Removal: The damaged fragment is removed by helicases and other proteins.
4. Gap Filling: DNA polymerase fills the gap using the undamaged strand as a template.
5. Ligation: DNA ligase seals the nick, restoring the integrity of the DNA strand.

Endonucleases in Mismatch Repair (MMR):

MMR corrects errors that occur during DNA replication, such as mismatched base pairs and small insertions/deletions. The MMR pathway involves the following steps:

1. Mismatch Recognition: MMR proteins recognize and bind to the mismatched base pair.
2. Strand Discrimination: The MMR system identifies the newly synthesized strand, which is more likely to contain the error.
3. Excision: An endonuclease cleaves the newly synthesized strand near the mismatch.
4. Exonuclease Digestion: An exonuclease removes the DNA segment containing the mismatch.
5. Gap Filling: DNA polymerase fills the gap using the template strand as a guide.
6. Ligation: DNA ligase seals the nick.

3. Exonucleases: Trimming DNA from the Ends

Exonucleases are enzymes that remove nucleotides from the ends of DNA strands. They play important roles in DNA replication, DNA repair, and DNA degradation. Exonucleases can be classified based on their directionality (5' to 3' or 3' to 5') and their substrate specificity (single-stranded or double-stranded DNA).

Types of Exonucleases:

• 5' to 3' Exonucleases: These enzymes remove nucleotides from the 5' end of a DNA strand. Examples include:

  • Exonuclease I: A single-stranded DNA exonuclease that removes nucleotides from the 5' end in a 3' to 5' direction.
  • Exonuclease T: A single-stranded DNA exonuclease that degrades DNA in the 5' to 3' direction.
  • DNA Polymerase I: In E. coli, DNA polymerase I has a 5' to 3' exonuclease activity that is used to remove RNA primers during DNA replication and to participate in DNA repair.

• 3' to 5' Exonucleases: These enzymes remove nucleotides from the 3' end of a DNA strand. Examples include:

  • Exonuclease III: A double-stranded DNA exonuclease that removes nucleotides from the 3' end in a 3' to 5' direction. It is specific for DNA containing nicks or gaps.
  • Proofreading Exonucleases: Many DNA polymerases have a 3' to 5' exonuclease activity that is used to proofread newly synthesized DNA. If the polymerase incorporates an incorrect nucleotide, the exonuclease activity removes it, allowing the polymerase to insert the correct nucleotide.

• Double-Stranded DNA Exonucleases: These enzymes degrade double-stranded DNA from either the 5' or 3' ends.

Exonucleases in DNA Repair:

Exonucleases play several roles in DNA repair pathways:

• Mismatch Repair: As mentioned earlier, exonucleases are involved in removing the DNA segment containing the mismatch in the MMR pathway.
• Base Excision Repair: In long-patch BER, an exonuclease may be involved in removing a longer stretch of DNA containing the AP site.
• Double-Strand Break Repair: Exonucleases are involved in processing DNA ends during homologous recombination repair of double-strand breaks. They can create single-stranded DNA overhangs that are necessary for strand invasion.

4. Flap Endonucleases (FEN1): Processing Flap Structures

Flap endonucleases (FEN1) are a class of structure-specific endonucleases that play critical roles in DNA replication and DNA repair. FEN1 enzymes recognize and cleave flap structures, which are single-stranded DNA segments that are displaced from a double-stranded DNA molecule.

How FEN1 Works:

FEN1 cleaves the phosphodiester bond at the base of the flap structure, removing the single-stranded DNA segment. This activity is essential for:

• Okazaki Fragment Processing: During lagging strand DNA replication, DNA is synthesized in short fragments called Okazaki fragments. These fragments are initiated by RNA primers, which must be removed before the fragments can be ligated together. FEN1 removes the RNA primers by cleaving the flap structure created when DNA polymerase displaces the primer.
• Long-Patch Base Excision Repair: In long-patch BER, DNA polymerase displaces a short segment of DNA while filling the gap created by the removal of the damaged base. FEN1 then removes the displaced flap structure.

The Importance of FEN1:

FEN1 is essential for maintaining genomic stability. Defects in FEN1 can lead to:

• Accumulation of Okazaki Fragments: If RNA primers are not efficiently removed, Okazaki fragments can accumulate, leading to replication stress and DNA damage.
• Defective DNA Repair: Impaired FEN1 activity can disrupt long-patch BER, leading to an accumulation of damaged bases in DNA.
• Increased Mutation Rates: Defects in FEN1 can increase mutation rates and genomic instability.
• Cancer Development: Mutations in FEN1 have been linked to an increased risk of cancer.

The Interplay of Enzymes in DNA Repair Pathways

The enzymes described above do not work in isolation. They function in a coordinated manner within complex DNA repair pathways. For example, in base excision repair, a DNA glycosylase initiates the process by removing a damaged base. This creates an AP site, which is then cleaved by an AP endonuclease. DNA polymerase fills the gap, and DNA ligase seals the nick.

Similarly, in nucleotide excision repair, endonucleases XPF-ERCC1 and XPG work together to make incisions on either side of the DNA lesion. Helicases remove the damaged fragment, DNA polymerase fills the gap, and DNA ligase seals the nick.

The coordinated action of these enzymes ensures that DNA damage is efficiently and accurately repaired, maintaining the integrity of the genome.

Implications for Human Health

The enzymes involved in DNA excision repair are essential for maintaining genomic stability and preventing disease. Defects in these enzymes can lead to:

• Increased Cancer Risk: Mutations in DNA repair genes are associated with an increased risk of various cancers. For example, mutations in BRCA1 and BRCA2, which are involved in double-strand break repair, are associated with an increased risk of breast and ovarian cancer.
• Premature Aging: Defects in DNA repair can lead to an accumulation of DNA damage, which can contribute to premature aging.
• Neurological Disorders: Some DNA repair genes are essential for neuronal development and function. Mutations in these genes can lead to neurological disorders.
• Immunodeficiency: DNA repair is also important for immune function. Defects in DNA repair can impair immune cell development and function, leading to immunodeficiency.

Therapeutic Potential

The enzymes involved in DNA excision repair are also potential targets for cancer therapy. For example, inhibitors of PARP (poly(ADP-ribose) polymerase), an enzyme involved in single-strand break repair, are used to treat cancers with defects in homologous recombination repair, such as those with BRCA1 or BRCA2 mutations.

By understanding the mechanisms of DNA excision repair and the roles of the enzymes involved, we can develop new strategies for preventing and treating diseases associated with DNA damage.

Conclusion

Enzymes that cut out damaged sections of DNA are essential for maintaining genomic stability and preventing disease. These enzymes function in a coordinated manner within complex DNA repair pathways to recognize, remove, and replace damaged DNA bases and nucleotides. Defects in these enzymes can lead to an increased risk of cancer, premature aging, neurological disorders, and immunodeficiency. Understanding the mechanisms of DNA excision repair is crucial for developing new strategies for preventing and treating diseases associated with DNA damage. The continued study of these remarkable molecular machines holds immense promise for improving human health.