Lecture 9

DNA Damage and Repair

• DNA replication cannot be perfectly accurate, as infinite precision requires infinite energy.
• Errors during DNA replication, without proofreading, result in 1 mistake per 10⁵ nucleotides copied.
• With proofreading, but without mismatch repair, errors decrease to 1 mistake per 10⁷ nucleotides copied.
• With mismatch repair, the error rate further reduces to 1 mistake per 10⁹ nucleotides copied.
• The Philadelphia chromosome is formed by translocation between chromosomes 9 and 22 [t(9;22)(q34;q11)], resulting in the formation of the Bcr-Abl fusion gene.
• The Abl gene is a proto-oncogene, and when combined with the Bcr gene it becomes an oncogene.
• The new protein, BCR-ABL, blocks DNA repair, impairs cells' ability to undergo apoptosis, and is created de novo in somatic cells.
• The Philadelphia chromosome occurs in 95% of chronic myeloid leukaemias, 25-30% of adults and about 6% in children with acute lymphoblastic leukaemias, and in less than 1% of acute myeloid leukaemias.

Mutation

• Mutation is a sudden, unintended change in a DNA nucleotide sequence.
• This arises due to the influence of mutagenic factors – both internal and external.
• Mutations alter genetic information in somatic and germ cells, which are passed onto daughter cells.
• Somatic mutations occur in body cells, not inherited, and may cause cancer.
• Germline mutations arise during gametogenesis or embryo development, may be inherited, leading to genetic diseases.
• De novo mutations occur in an organism but not its parents. They can arise spontaneously or from induced factors, affecting somatic and germline cells, and sometimes the embryo itself.

Spontaneous Mutations

• Spontaneous mutations can emerge from errors during DNA replication and transcription.
• Replication errors are linked to modifications or loss of nitrogenous bases in DNA, accompanied by DNA polymerase slippage.
• Transcription errors are tied to changes in promoter or regulatory sequences, alongside alternative splicing.

Base Modifications

• Spontaneous base modifications in DNA can stem from various sources, including tautomerization, oxidative deamination, oxidation, methylation, and depurination/depyrimidination.

Base Tautomerization

• Nitrogen bases exist in tautomeric forms, structural isomers differing in proton and double bond positions.
• Examples include the keto/enol forms of thymine/uracil/guanine and amino/imine forms of adenine/cytosine.
• Rare tautomeric forms can contribute to incorrect base pairings, resulting in mutations.

Oxidative Deamination

• Oxidative deamination replaces an amino group (-NH₂) with an oxygen atom (-O-).
• This leads to conversions: adenine to hypoxanthine, guanine to xanthine, and cytosine to uracil.
• These conversions can cause mutations by altering correct base pairings.

Oxidation

• Guanine reacting with reactive oxygen species (ROS) forms 8-oxoguanine (8-oxoG).
• 8-oxoG pairs with adenine rather than guanine, leading to G:C to 8-oxoG:A mutations, a common spontaneous DNA damage.

Depurination and Depyrimidination

• Depurination removes purine bases from a nucleotide, while depyrimidination removes pyrimidines.
• A hydroxyl group (-OH) takes the place of the removed base, causing significant DNA damage.
• Depurination is more common than depyrimidination.

Replication Slippage

• DNA polymerase can make errors due to "slippage errors," leading to fragments of DNA being duplicated.
• Expansion/contraction typically involves trinucleotide repeats, with duplication lengths of two, three, four, or occasionally five nucleotides.
• This mostly affects the lagging strand.

Transcriptional Mutations

• Mutations in promoter, enhancer, or silencer sequences can affect gene transcription levels impacting mRNA production and protein levels.
• Either decreasing or increasing gene transcription levels in this way, can have drastic effects on the levels of proteins in a cell.

Splicing Mutations

• During post-transcriptional processing, mutations in splicing, including exon skipping, exon shuffling, and intron retention can alter the nucleotide sequence in mature mRNA and therefore the amino acid sequence in proteins.

Mutagenic Factors

• Physical mutagens include ionizing radiation (e.g., alpha, beta, gamma rays, X-rays), and non-ionizing radiation (e.g., ultraviolet radiation).
• Chemical mutagens are categorized as base analogs, chemicals, or intercalators, which exert their effects by altering the structure and properties of DNA.
• Biological mutagens include viruses and bacteria.

Cell Response to Mutation

• Cells can repair damage, die (apoptosis), or pass on mutations to daughter cells. This encompasses partial DNA repair or complete restoration to the original DNA structure, or the complete deletion of a cell to prevent further damage.

Influence of Mutations on Phenotype

• Mutations can be beneficial, inert, adverse, or lethal, impacting the organism's traits (phenotype).

Mutation Types

• Point mutations are alterations to the DNA's molecular level.
• These can include substitution, insertion, deletion, inversion, and duplication of nucleotides within the DNA sequence.
• Chromosomal mutations affect chromosome structure or numbers.
• These could involve deletions, insertions, inversions, duplications, translocations (reciprocal or non-reciprocal), Robertsonian translocations, centric division, isochromosomes, ring chromosomes, and dicentric chromosomes.
• Beyond chromosomal, mutations can occur in mitochondria or chloroplasts.

Point Mutations

• These modify the specific nucleotide sequence, sometimes leading to incorrect and dysfunctional proteins.

Types of Point Mutations

• Includes substitutions (transition or transversion), insertions, deletions, inversions and duplication.

Effects of Point Mutations

• Silent mutations have no impact on the encoded amino acid.
• Nonsense mutations premature create a STOP codon.
• Missense mutations change the encoded amino acid, potentially affecting protein function (conservative or non-conservative).
• Frameshift mutations alter the reading frame, affecting all subsequent codons.

Silent Mutations

• Nucleotide changes in the third codon position often do not alter the amino acid encoded, or cause no change in the protein encoded.
•   These are often considered polymorphisms rather than mutations when occurring in more than 1% of a population (SNP).

Nonsense Mutations

• Produce premature STOP codons, shortening the protein and leading to dysfunction.

Missense Mutations

•   Altering the encoded amino acid could lead to conservative or non-conservative changes impacting a protein's function.

Frameshift Mutations

•   Insertion or deletion of one or more nucleotides that are not evenly divisible by 3 causes a shift in the reading frame, drastically alters the amino acid sequence, and creates a dysfunctional protein.

DNA Repair

• Cells have mechanisms to protect their genome from altered structure.
• DNA damage responses typically occur in three stages - Recognition, Repair, and Restoration.
• Stage I: damaged DNA is recognized and removed via enzymes called nucleases, which cut phosphodiester bonds connecting DNA nucleotides in the damaged strand.
• Stage II: A DNA polymerase will utilize the undamaged DNA strand as a template to synthesize a copy of the complementary strand.
• Stage III: the breaks in the sugar-phosphate backbone of the repaired DNA strand are then joined together to complete the repair process using another DNA enzyme, DNA ligase.

DNA Repair Systems

• Direct repair involves repairing mismatched nucleotides.
• Excision repair manages damage to single-stranded DNA (base excision repair or nucleotide excision repair).
• Double-stranded DNA breaks repair utilizing two distinct mechanisms (nonhomologous end joining and homologous recombination).

Direct Repair

• Involves removing alkyl groups from specific DNA bases without breaking the DNA strand's continuity. Methyltransferase enzymes achieve this removal of potentially harmful alkyl groups.

MMR-type Repair

• Corrects mismatched nucleotides using specific proteins (MutS, MutL, MutH).

Base Excision Repair

• Specialized enzymes, DNA glycosylases remove damaged nitrogenous bases creating AP sites. 

Nucleotide Excision Repair

• Repairing larger DNA damage using complex proteins (XPA, XPC, XPG, XPF).

Double Strand DNA Break Repair

•   Non-homologous end joining (NHEJ) and homologous recombination (HR) repair double-stranded breaks in DNA accurately, though NHEJ may introduce errors.

NHEJ Repair

•   Ku proteins recognize broken ends, and other proteins like Artemis and endonuclease remove damaged parts.
•   The ends are then joined by the XRCC4-ligase complex, resulting in possible nucleotide deletions.

HR Repair

• Initiated immediately after replication.  ATM kinase identifies the break and triggers cell-cycle arrest. A single strand is then formed.
• A homologous, undamaged DNA strand from the sister chromatid is used as a template to repair the damaged site.

Chromosomal Aberrations

• These are structural or numerical mutations large enough to be observed under a microscope.
• Structural aberrations involve changes in chromosome structure (deletions, insertions, inversions, duplications, translocations).
•   Numerical aberrations involve changes in the number of chromosomes (aneuploidy and polyploidy).
•   Causes of aneuploidy - Nondisjunction (failure of chromosome separation during meiosis or mitosis) is a common cause.