DNA Damage and Repair Mechanisms

DNA Damage and Repair

• DNA damage can occur due to various factors, including the release of aromatic bases, oxidative damage to bases, crosslinking of bases, and breaks in phospho-ester strands.
• Humans have 150 genes that are responsible for DNA repair and maintaining genetic stability.

Proof-Reading and Mismatch Repair

• DNA polymerases have high fidelity and are accurate, with an error rate of 1 wrong nucleotide per 10^8-10^9 nucleotides.
• DNA polymerases have 3'-5' exonuclease proof-reading activity, which corrects misincorporation.
• Mismatch repair proteins act if proof-reading fails, correcting mismatches.

Translesion Synthesis

• Translesion synthesis (TLS) pols can insert residues opposite damaged sites, allowing DNA replication to proceed.
• TLS pols lack proof-reading, increasing the chance of mistakes.
• The recruitment of TLS pols is coordinated by the sliding clamp PCNA, which swaps pols at the 3'-end of the DNA strand.

Trinucleotide Repeat Diseases

• Short tandem repeats can display polymorphism, leading to errors in replication.
• Trinucleotide repeat diseases occur when the expansion of repeats impairs the function or behavior of a gene product.
• Huntington's disease is an example of a trinucleotide repeat disease, caused by the expansion of CAG repeats in the Huntingtin gene.

UV Light-Induced Damage

• UV light can crosslink adjacent thymine residues, forming cyclobutyl ring dimers that deform the double helix and obstruct DNA replication and RNA transcription.
• This can be reversed in bacteria by DNA photolyases, which use light to split the dimer.

Deamination and Base Excision Repair

• Deamination of cytosine to uracil can convert CG to TA in later generations.
• DNA glycosylase recognizes uracil in DNA and flips it out of the double helix, cleaving the glycosidic link to deoxyribose and leaving an abasic site.
• This site is recognized by a nuclease, which cleaves the backbone and produces a 3'OH upstream of the abasic site, acting as a priming site for DNA pol to fill in.

Oxidative Damage and Mismatch Repair

• Oxidative damage can form 8-oxoGuanine, leading to GC to TA transversions if unrepaired.
• A specific glycosylase removes oxidized bases.

Methylation and Gene Expression

• Methylation of DNA on C/A by Dam methyltransferase controls gene expression in bacteria.
• In eukaryotes, methylation occurs at CpG islands to switch off nearby genes, which is the basis of genomic imprinting.

Nucleotide Excision Repair

• Nucleotide excision repair (NER) repairs pyrimidine dimers and distortions of the double helix in eukaryotes and prokaryotes.
• NER exists as global genomic and transcription-coupled NER, which differ in proteins but have the same steps.

Double-Strand Breaks

• Double-strand breaks (DSBs) can be caused by radiation, reactive oxygen species, and during replication, leading to cell death or high mutagenicity.
• There are two main repair mechanisms for DSBs: non-homologous end joining (NHEJ) and homologous recombination (HR).

Non-Homologous End Joining

• NHEJ sticks two broken ends of DNA together, restoring the original sequence, but can be inherently mutagenic.
• NHEJ is important in resting G1 and G0 phases.

Homologous Recombination

• Homologous recombination is a more complex but potentially error-free mechanism that uses an extra copy of the chromosome as a template for repair.
• The process generates Holliday junctions, which can slide up and down the DNA.

DNA Repair and Cancer

• Loss of NER protein function and DSB protein increases cancer predisposition.
• Mutations in BRCA2, ATM, Chk2, p53, Nbs1, and Mre11 increase cancer risk.
• Some cancers resist radiation by increasing their ability to repair radiation-induced breaks.

Synthetic Lethality

• Inhibiting specific DNA repair pathways used by cancer cells can have little impact on the rest of the cell, a concept known as synthetic lethality.
• Examples include PARP inhibitors for BRCA2-deficient cancers.

DNA Polymerase Accuracy

• DNA polymerases are highly accurate, with an error rate of approximately 1 incorrect nucleotide per 108-109 nucleotides.
• This accuracy is due to the 3'-5' exonuclease proof-reading activity of DNA polymerases, which allows them to correct errors during DNA replication.
• In cases where proof-reading fails, Mismatch Repair (MMR) proteins can step in to correct errors.
• Mutations in the proof-reading domain of DNA polymerase epsilon have been implicated in driving cancer formation through hyper-mutation.

DNA Translesion Synthesis (TLS)

• TLS polymerases (Pols) insert residues opposite damaged sites, allowing DNA replication to proceed despite the presence of unrepaired DNA damage.
• TLS Pols lack proof-reading activity, which increases the chance of mistakes during DNA replication.
• The recruitment of TLS Pols is coordinated by the Sliding Clamp PCNA, which facilitates the swapping of Pols at the 3’-end of the DNA strand.

Trinucleotide Repeat Diseases

• Trinucleotide repeat diseases occur when the expansion of repeats impairs the function or behavior of the gene product.
• Repeat expansion can cause errors in replication, making some repeats more prone to errors.

Huntington's Disease

• Huntington's disease is caused by CAG repeats in the Huntingtin gene.
• Individuals with fewer than 30 repeats do not develop the disease.
• Individuals with over 40 repeats will develop the disease.
• Huntington's disease demonstrates anticipation, where affected offspring develop the condition earlier due to an increased number of repeats.
• The increase in repeat number is due to the transient hairpin formation in the repeat regions, causing the machinery to expand the region.

Effects of UV Light on DNA

• UV light can cause crosslinking of adjacent thymine residues, forming cyclobutyl ring dimers, which deform the double helix and obstruct DNA replication and RNA transcription

DNA Repair Mechanisms

• DNA photolyases can reverse thymine dimers using light
• Deamination of cytosine to uracil can lead to CG to TA conversion in later generations, known as a transition
• Uracil in DNA is recognized by DNA glycosylase (UDG), which flips it out of the double helix, cleaves the glycosidic link, and leaves an abasic site
• Abasic site is recognized by a nuclease, which cleaves the backbone, producing a 3'OH upstream of the abasic site, acting as a priming site for DNA polymerase to fill in

Base Excision Repair (BER)

• BER can occur via Long-Patch BER or Short-Patch BER
• Long-Patch BER involves the same proteins that process Okazaki fragments synthesizing over the abasic site
• Short-Patch BER involves specialized DNA repair polymerase beta filling in the gap
• DNA ligase seals the nick after repair

Oxidative Damage and DNA Repair

• Oxidative damage can form 8-oxoGuanine, leading to GC to TA transversion, commonly seen in cancers
• A specific glycosylase removes the oxidized base to prevent mutations

DNA Methylation and Gene Expression

• DNA methylation by DAM methyltransferase (in bacteria) controls gene expression and protects DNA from digestion by restriction endonucleases
• In eukaryotes, DNA methylation occurs at CpG islands, switching off nearby genes, which is the basis of genomic imprinting
• Genomic imprinting results in parent-of-origin expression, where one copy of the gene is silent due to methylation

Nucleotide Excision Repair (NER)

• Repairs pyrimidine dimers and distortions of the double helix in both eukaryotes and prokaryotes.
• The NER process involves the detection of a lesion, followed by the binding of protein machinery, unwinding of DNA, cleavage of the strand containing the lesion, and the creation of a short ssDNA gap.
• The ssDNA gap is then filled by DNA polymerase.

Global Genomic NER and Transcription-Coupled NER

• Both types of NER follow the same steps, but differ in the proteins involved.

Xeroderma Pigmentosum (XP)

• A genetic disorder caused by mutations in genes encoding NER enzymes.
• Characterized by autosomal recessive inheritance.

Methyl-Directed Mismatch Repair (MMR) in Bacteria

• Bacterial DNA is typically methylated, allowing for the identification of non-methylated DNA.
• Non-methylated DNA is excised, and the resulting gap is filled by DNA polymerase.

MMR System Defects and Cancer

• Mutations in the MMR system are associated with a predisposition to colorectal cancer.

DNA Repair

• Sticking two broken ends of DNA together can restore the original sequence, but often requires cleaning, resulting in loss of DNA at the joining site, making it inherently mutagenic.

Non-Homologous End Joining (NHEJ)

• NHEJ is crucial in the resting G1 and G0 phases of the cell cycle.

NHEJ and Cancer

• Mutations in NHEJ proteins can lead to cancer, such as mutations in DNA ligase IV, which are associated with certain types of leukemia.

Homologous Recombination (HR)

• HR is a complex but potentially error-free mechanism for repairing damaged DNA.
• It uses an extra copy of the chromosome as a template for repair.

Process of HR

• dsDNA break is processed, trimming the 5’ end and leaving an extended ssDNA 3’ end.
• The ssDNA end is recognized by RecA (in bacteria) or RAD51 (in eukaryotes).
• RecA/RAD51 scans the genome for identical dsDNA sequences.
• Once a suitable target is found, it directs strand invasion, replacing one duplex strand with the ssDNA damaged strand.
• The invaded end is primed for replication, copying DNA from the intact strand to fill the gap.

Holliday Junctions

• The process generates Holliday Junctions, which are mobile four-way junctions in DNA.
• Holliday Junctions can slide up and down the DNA.
• In bacteria, the sliding is facilitated by the RubAB complex, which uses ATP.

Outcomes of HR

• HR can restore two linear duplexes to regenerate parental DNA.
• Alternatively, HR can lead to a crossed-over configuration (where DNA is swapped), which occurs in meiosis.
• HR also restarts stalled or broken replication forks, which is essential for cell division (10 forks per eukaryotic cell cycle must be resolved).

DSB Repair and Cancer

• Loss of NER protein function and DSB protein can lead to cancer predisposition.
• BRCA2 mutations are associated with sporadic and inherited breast cancer and play a role in Homologous Recombination (HR) repair.
• Mutations in ATM, Chk2, p53, Nbs1, and Mre11, which are involved in DSB repair, increase cancer risk.

Radiation Resistance and Cancer

• Some brain tumors develop resistance to radiation by increasing their ability to repair radiation-induced DNA breaks.

Positive and Negative Consequences of DSB Repair

• DSB repair can be beneficial, such as in generating antibody diversity by joining different DNA segments, increasing variation.
• However, DSB repair can also be detrimental, such as in the translocation of parts of chromosome 9 and 22, leading to Chronic Myelogenous Leukaemia.

Antibody Diversity Generation

• Enzymes like Terminal Transferase add extra nucleotides at junctions, increasing diversity by being sloppy in the number of bases added.
• Non-Homologous End Joining (NHEJ) mediates antibody diversity generation, and mutations in NHEJ genes lead to immunodeficiency and failure to perform antibody diversity generation.

Cancer Repair Mechanisms

• Cancer cells inactivate certain repair mechanisms to gain a survival advantage.
• Specific pathways are exploited by cancers more frequently, making them potential targets for inhibition.

Synthetic Lethality

• Synthetic lethality is a strategy to inhibit cancer-specific pathways with minimal impact on normal cells.
• This approach is achieved by targeting specific pathways that are critical for cancer cell survival.

PARP Inhibitors

• Olaparib is a PARP (Poly ADP-ribose polymerase) inhibitor.
• PARP inhibitors are effective against cancer cells with BRCA2 defect genes.
• BRCA2 defect genes are associated with increased cancer susceptibility.