DNA damage and repair
Vast majority of DNA compromises of non-coding DNA made of regulatory sequences
Whole collection of alleles – genotype aka genetic makeup
What produces genetic variation?
Mutation
Mitochondrial DNA
Inherited and affects protein production/function
-SNPs have one base pair/nucleotide varied in different individuals of population (2 million per genome)
Point mutation is the change in a single nucleotide in the DNA sequence that can nucleotide substitutions, insertions or deletions. Point mutations are more rare compared to SNP.
DNA polymerase maintain low error rate during DNA replication
-Base pairing mismatch: DNA polymerase relies on complementary base pairing. If the template strand has an unusal base or if the polymerases makes a mistake in selecting nucleotide, an incorrect nucleotide can be added.
Proofreading will remove the incorrect nucleotide inserted by 3’ to 5’
IDLs (Insertion/Deletion Loops) are small insertions or deletions in DNA sequences that can form during DNA replication. When these loops go uncorrected, they can lead to mutations, potentially affecting the function of genes or regulatory elements.
Replication Slippage is a common mechanism behind IDL formation. During DNA replication, the DNA polymerase may temporarily lose its place, causing it to “slip” on the DNA strand. This slippage often occurs in regions with repetitive sequences, where the DNA strand can misalign with itself. Depending on how the strand realigns, the result can be an insertion or deletion in the new DNA strand once replication resumes.
Spontaneous mutations are changes in DNA sequence that occur naturally without external influence. Eg. Mistakes made by DNA polymerase or chemical instability such as DNA bases undergoing deamination so an amine group is removed which alters DNA sequence.
Induced mutations are genetic changes caused by external factors, often referred to as mutagens. These mutagens can be physical, like radiation (e.g., UV or X-rays), or chemical, such as certain carcinogens. Unlike spontaneous mutations, which occur naturally during DNA replication, induced mutations happen because an outside agent actively damages or alters the DNA structure.
Mutagens can cause various types of mutations. For instance, UV radiation can lead to thymine dimers, where two adjacent thymine bases bond together, disrupting DNA replication. Chemical mutagens may alter DNA bases, leading to mispairing during replication. If the DNA damage is not properly repaired, these changes can become permanent, potentially leading to disruptions in gene function or regulation, which may contribute to diseases like cancer.
A base substitution or point mutation occurs when one base in a DNA sequence is replaced by another. These substitutions can be categorized into two main types: transitions and transversions.
• Transitions happen when a purine (adenine or guanine) replaces another purine, or a pyrimidine (cytosine or thymine) replaces another pyrimidine. These are generally more common because the similar structure of the bases makes them easier for DNA repair mechanisms to miss.
• Transversions occur when a purine is substituted for a pyrimidine, or vice versa. This type of mutation is less common but can have a more significant impact on the DNA structure, as it swaps a single-ringed base for a double-ringed one (or the opposite), leading to greater structural disruption.
Sickle cell disease is caused by a transversion mutation in the hemoglobin gene. This mutation swaps one DNA base for another, changing a glutamic acid to a valine in the hemoglobin protein. This small change makes red blood cells take on a stiff, sickle shape, which can block blood flow and lead to pain and other symptoms of the disease.
Silent mutation – nucleotide change occurs in the DNA sequence but it does not alter the AA sequence of the resulting protein. Occurs due to redundancy in the genetic code where multiple codons can code for the same AA. This can affect gene expression/slicing instead.
Missense Mutation:
Definition: A missense mutation is a type of point mutation where a single nucleotide change results in a codon that codes for a different amino acid. This alteration leads to the incorporation of an incorrect amino acid into the protein sequence during translation.
Types:
Conservative Missense Mutation: Here, the new amino acid has similar properties (like charge, hydrophobicity) to the original, potentially leading to a less dramatic change in protein function.
Non-Conservative Missense Mutation: The new amino acid differs significantly in properties from the original, which might lead to a more pronounced effect on the protein's structure or function.
Nonsense Mutation:
Definition: A nonsense mutation occurs when a point mutation changes a codon that would code for an amino acid into one of the three stop codons (UAG, UGA, UAA). This results in the premature termination of translation, leading to a truncated protein.
Effect: The resulting protein is shorter than normal because translation stops early, often leading to loss of function or significant alteration in protein activity due to missing essential domains.
remember-A mutation that changes a part of the DNA code meant to be an amino acid into a stop signal, which tells the cell to stop making the protein too early.
Insertion, Deletion, and Frameshift Mutations:
Insertion and Deletion:
Insertion: An insertion mutation occurs when extra base pairs are added into the DNA sequence.
Deletion: A deletion mutation happens when base pairs are removed from the DNA sequence.
Frameshift Mutation:
Definition: Frameshift mutations occur when insertions or deletions are not in multiples of three base pairs. Since codons are read in groups of three, adding or removing bases in numbers not divisible by three shifts the reading frame.
Effect: This shift changes how the DNA sequence is divided into codons, altering the sequence of amino acids from the point of mutation onward. This can lead to a completely different translation downstream of the mutation, potentially resulting in a protein with different or no function.
What: This happens when you add or remove DNA letters but not in groups of three, which messes up how the DNA message is read into proteins. This can lead to:
Insertion: Adding extra DNA letters.
Deletion: Removing DNA letters.
Key Point: Imagine shifting all the words in a sentence by one or two letters; it changes the whole meaning, like shifting the reading frame of the DNA changes the protein sequence.
Example:
Original sequence: ATG GGC TCA (coding for methionine-glycine-serine)
Insertion: Adding one nucleotide (A): ATG AGG CTC A...
The reading frame is shifted, resulting in different amino acids being coded.
Deletion: Removing one nucleotide (G): ATG GCT CA...
Again, the reading frame shifts, leading to an altered protein sequence.
Functional Consequences of Mutations (Mutant Protein)
Back Mutation or Reversion:
What: A mutation that restores the DNA to its original sequence or function, bringing back the wild type phenotype (normal function).
Key: It's like hitting the "undo" button on a previous mutation, reverting to the normal, or wild type, state.
Conditional Mutations:
What: These mutations cause a change in protein function only under certain conditions. For example, a protein might work normally at a lower temperature (25°C) but fail at a higher one (37°C).
Key: The wild type phenotype is only altered under specific environmental conditions, showing the mutated phenotype (altered function) otherwise.
Lethal Mutations:
What: Mutations so detrimental that they lead to the death of the organism, meaning the wild type phenotype cannot be observed because the organism doesn't survive to display it.
Loss of Function Mutations:
What: These mutations result in a protein that either doesn't work at all or works poorly, leading to a loss of the wild type phenotype.
Types:
Point Mutation: A single nucleotide change.
Truncation: The protein is shorter than intended.
Deletion: Part of the gene or protein is missing.
Genetic Impact: Often recessive, meaning two copies of the mutation are needed for the mutated phenotype to be evident, as one functioning copy (from the other parent) might be enough to maintain the wild type phenotype.
Gain of Function Mutations:
What: Mutations where the protein acquires a new or enhanced function, leading to a mutated phenotype that's different or stronger than the wild type phenotype.
Genetic Impact: These mutations are often dominant, as even one copy can alter the protein's function enough to manifest a new trait.
Somatic Occur in: Somatic cells, which are all the cells in the body except for sperm and egg cells (e.g., skin, liver, or muscle cells).
Inheritance: Not passed on to offspring because they do not affect the reproductive cells.
Impact: Affects only the individual in whom the mutation occurs. The mutation can lead to diseases like cancer if it happens in a gene that controls cell growth.
Examples: Skin cancer caused by UV radiation, or lung cancer due to smoking, are often the result of somatic mutations in specific cells.
Germline: Occur in: Germ cells, which are the cells that give rise to sperm and eggs.
Inheritance: Can be passed on to offspring, meaning the mutation is present in every cell of the child's body.
Impact: Affects not just the individual with the mutation but can be transmitted across generations, potentially causing inherited genetic disorders.
Examples: Conditions like cystic fibrosis, hemophilia, or Huntington's disease are caused by germline mutations in specific genes.
DNA Repair Pathways
Overview:
DNA can be damaged by various sources like ionizing radiation, chemicals, UV light, and replication errors.
The body uses specific pathways to repair these damages, categorized by the type of damage.
Types of Damage and Repair Pathways:
Non-bulky DNA Lesions:
Damage Source: Chemicals, Ionizing Radiation.
Repair Pathway: BER (Base Excision Repair).
Process: Enzymes recognize and remove the non-bulky lesions (like oxidized bases) and replace them with the correct nucleotides.
Double Strand Breaks:
Damage Source: Ionizing Radiation, Chemicals.
Repair Pathway: HR (Homologous Recombination) or NHEJ (Non-Homologous End Joining).
HR Process: Uses a sister chromatid as a template to repair the break accurately.
NHEJ Process: Directly joins the broken ends, which might lead to small insertions or deletions, not always accurate.
Bulky DNA Lesions:
Damage Source: UV light, Chemicals.
Repair Pathway: NER (Nucleotide Excision Repair).
Process: Removes large distortions in the DNA structure (like thymine dimers from UV light) and fills in the gap with correct nucleotides.
Mismatched Bases:
Damage Source: Replication Errors.
Repair Pathway: MMR (Mismatch Repair).
Process: Corrects errors that occur during DNA replication where incorrect bases are paired. It scans the newly synthesized DNA strand and corrects mismatches.
Repair Strategy for Single Strand Damage
Steps for Repairing Single Strand Damage:
Damage Recognition:
Objective: Identify and locate the damaged part of the DNA strand.
Excision of Damaged Part:
Process: Enzymes cut out the section of the DNA containing the damage, creating a gap.
Replacement of Damaged Part:
Step 1: The damaged segment is removed.
Step 2: DNA Polymerase uses the undamaged strand as a template to fill in the missing nucleotides.
Step 3: DNA Ligase seals the new segment into the backbone of the DNA strand, restoring its integrity.
Simplified Explanation:
DNA gets damaged from things like sunlight, chemicals, or when cells copy their DNA.
Our body has repair teams (like BER, NER, MMR, HR, NHEJ) that fix these damages.
For minor damage, like a single strand issue, the process involves:
Finding the damage,
Cutting it out,
Filling the gap with the right pieces using the other strand as a guide,
Sealing it up to make the DNA whole again.
1. Base Excision Repair (BER)
Purpose: This repair pathway corrects non-bulky lesions such as abasic sites, oxidation, deamination (like conversion of cytosine to uracil), or single-strand breaks.
Steps:
Recognition & Removal: A specific DNA glycosylase enzyme recognizes the damaged or inappropriate base. It flips the base out of the DNA helix and cleaves the bond connecting it to the sugar-phosphate backbone, creating an abasic site.
Incision: An AP endonuclease (like AP endonuclease or DNA-(apurinic or apyrimidinic site) lyase) makes an incision at the abasic site, breaking the phosphodiester backbone of the DNA.
Gap Filling & Sealing: The gap is filled in by a DNA polymerase, which synthesizes new DNA using the intact strand as a template. The newly synthesized strand is then sealed to the existing strand with a phosphodiester bond by DNA ligase, restoring the DNA backbone continuity.
2. Nucleotide Excision Repair (NER)
Purpose: NER is specialized for repairing bulky, helix-distorting lesions like thymine-thymine dimers caused by UV radiation or large chemical adducts.
Types: There are two sub-pathways:
Global Genomic NER (GG-NER): Repairs damage anywhere in the genome.
Transcription-Coupled NER (TC-NER): Focuses on lesions that block RNA polymerase during transcription.
Steps:
Damage Detection: A protein complex detects the distortion in the DNA helix.
Unwinding: Another protein complex unwinds the DNA around the damage site, creating a 'bubble' of about 25 bases.
Incision: Endonucleases cut the damaged strand at positions approximately 24-32 bases apart, releasing a segment of single-stranded DNA containing the lesion.
Excision & Repair: The damaged DNA strand is removed, and DNA polymerase fills in the gap using the complementary strand as a template. DNA ligase then seals the new strand into the DNA backbone with phosphodiester bonds.
3. Mismatch Repair (MMR)
Purpose: MMR corrects errors that occur during DNA replication, such as base-pair mismatches or small insertions/deletions (indels) due to replication slippage.
Mechanism:
Mismatch Recognition: A protein complex (in eukaryotes, involving proteins like MutS homologs) recognizes and binds to the mismatch.
Strand Discrimination: The system identifies which strand is the newly synthesized one (often through strand-specific methylation patterns).
Incision & Removal: An endonuclease (like MutL homologs in eukaryotes) makes a cut in the new strand, and an exonuclease removes the segment containing the mismatch.
Resynthesis & Sealing: DNA polymerase synthesizes the correct sequence using the old strand as a template, and DNA ligase seals the corrected strand back into the DNA backbone with phosphodiester bonds.
Homologous Recombination (HR)
What it does: HR repairs major breaks in DNA by using a similar piece of DNA, usually from the sister chromatid, as a guide for accurate repair.
Steps:
Break Recognition: Proteins spot the break, creating single-stranded 3' overhangs.
Strand Invasion: RAD51 proteins bind to these overhangs, facilitating the invasion of one strand into the homologous DNA (from the sister chromatid), forming a D-loop.
Repair and Resolution: Using this D-loop, the DNA is repaired:
Synthesis-Dependent Strand Annealing (SDSA): New DNA is synthesized based on the template, then the invading strand reconnects with its original partner.
Double Holliday Junctions (dHJ): Complex structures might form, which are then resolved, potentially leading to genetic exchange.
Why it's Good: HR is very accurate because it uses a template, like copying from the correct page of a book.
Non-Homologous End Joining (NHEJ)
What it does: NHEJ quickly fixes DNA breaks by directly joining the ends, which might introduce small errors.
Steps:
End Binding: Proteins like Ku70/Ku80 bind to the broken ends right away.
Synapsis Formation: A 'synapse' brings the broken ends together. Here:
End Processing: Nucleases might:
Trim: Cut away extra or damaged nucleotides to prepare the ends.
Polymerases: Add nucleotides to fill gaps or make the ends compatible, which could lead to small insertions or deletions (indels).
Ligation: Enzymes like Ligase IV and XRCC4 seal the ends with new bonds, but without a template, the original sequence might not be exactly restored. Sometimes, if the break is at the same chromosome locus, ends from different chromosomes might accidentally join.
Same Chromosome Locus: This means the break occurs at a similar position or region on two different chromosomes.
Accidental Joining: NHEJ doesn't use a template for repair, so it can sometimes mistakenly join broken ends from different chromosomes if they happen to be at the same location or close enough in spatial terms within the cell nucleus. This can happen because NHEJ primarily focuses on quickly sealing the break rather than ensuring the correct sequence or chromosome is used.
Key Differences:
Accuracy: HR is precise, like following a blueprint; NHEJ is less so, like guessing how to fix a puzzle.
Speed: NHEJ is faster since it doesn't need to search for a matching DNA piece.
Outcome: HR aims to keep the original sequence; NHEJ might alter it slightly due to the action of nucleases and polymerases, especially if the break occurs at the same chromosome locus where ends from different chromosomes might be mistakenly joined.
In summary, HR uses a sister chromatid for a precise repair, while NHEJ quickly joins the ends, potentially changing the DNA with the help of nucleases and polymerases, and can lead to mistakes if the break is at the same chromosome locus.