DNA Strands and Inheritance of Mutations

• Mutation refers to a gene that has a different sequence compared to the normal or wild type gene
• Mutations can be inherited, with a variety of effects on the phenotype
• The majority of mutations are neutral, with no effect on the organism

DNA Strands and Mutations

• Two DNA strands are polynucleotides, composed of monomeric units called nucleotides
• A nucleotide is composed of one of four nitrogen-containing nucleobases: cytosine (C), guanine (G), adenine (A), or thymine (T)
• Nucleobases also include a sugar called deoxyribose, and a phosphate group
• Nucleotides are joined in a chain by covalent bonds
• More than 98% of human DNA is non-coding, thus these sections do not serve as patterns for protein sequences
• Genetic information is encoded in the sequence of the four nucleobases along the backbone

Inheritance of Mutations

• A mutation must be present in the germline cells (meiotic) to be inheritable in multicellular organisms
• Mutations in somatic cells (mitotic) are not inherited
• Changes to the RNA code, or errors in transcription, are not inherited

Static vs Dynamic Mutations

• Static mutations become stably incorporated into the genome of germline and somatic cells, excluding red blood cells (RBCs)
• Static mutations are transferred to the next generation
• The offspring's genome is the same as the parent such as sickle cell anemia
• A phenotype's ultimate expression depends on genetic information from both parents and epigenetic factors
• Dynamic mutations are illustrated by trinucleotide repeats (TNR)
• Dynamic mutations increase in severity with each generation, increasing the number of repeats
• Dynamic mutations vary between tissues of the same organism
• Subsequent generations are more affected by dynamic mutations
• An increase in copy number of the repeat occurs at replication, repair, or recombination
• When DNA is copied, TNR repeat number growth leads to a growing polyglutamine tract that produces an HD gene product (huntingtin) which has aberrant properties that cause death of brain cells

Types of Mutations

• Transversion occurs when a purine is replaced by a pyrimidine, or vice versa
• Transition occurs when a pyrimidine is replaced by a pyrimidine, or a purine for a purine
• Silent mutations involve altered codons that still code for the same amino acid
• Frameshift mutations shift the reading frame through added or deleting bases, leading to a non-functional protein
• Neutral mutations involve altered codons coding for a functional similar amino acid, thus, having no affect on protein functionality
• Point mutations change a single base pair through a substitution, deletion, or addition
• Missense mutations involve altered codons for functionally different amino acids and may be lethal
• Nonsense mutations produce a stop codon, leading to truncated proteins which can be also dangerous
• Splice mutations produce or remove a splice site; only occurs in eukaryotes
• Temperature sensitive mutations change the protein function so it is temperature sensitive
• Usually, the protein functions normally at lower permissible temperatures (<30°C) but is inactive at higher temperatures (>40°C)
• Leaky mutations do not affect the organism under normal conditions, but they will show up in "stressed" conditions

Mutagenesis

• Mutagen is a physical or chemical agent that causes mutation to occur at a higher frequency
• Natural or spontaneous mutations occur at a normal background rate all the time
• These mutations from the genome arise naturally in the course of a cell's life

Mechanisms of Induction of Genetic Alterations

• DNA damage includes single- and double-strand breaks in the DNA backbone
• DNA damage also includes cross-links between DNA bases, between DNA bases and proteins as well as chemical addition to the DNA bases in the form of adducts
• Ionizing radiations produce DNA single- and double-strand breaks and a broad range of base damages though the proportions vary with the type of radiation
• Ultraviolet(UV) light is nonionizing radiation that induces two predominant lesions: cyclobutane pyrimidine dimers and 6,4-photoproducts and blocks polymerization by DNA polymerase I
• Chemicals produce base alterations either directly as adducts or indirectly through the intercalation of a chemical between the base pairs
• Many electrophilic chemicals react with DNA, forming covalent addition products (adducts)
• Alkylated bases causes base loss from the DNA AP site which causes mutations by insertion of incorrect bases into AP sites

Endogenous Agents

• Endogenous agents are responsible for several hundred DNA damages per cell per day
• Cellular processes leading to DNA damage are the formation of reactive active oxygen species and deamination of cytosines and S-methylcytosines
• Deamination leads to uracils and thymines, respectively
• DNA replication is error-prone, thus an incorrect base can be added by the polymerase

Other Mutagenic Agents

• Intercalators which are planar ring structures slide in between the base pairs causing a disruption to the normal base stacking like ethidium Bromide, acridine orange, actinomycin D
• Alkylating agents methylate or ethylate bases, which results in altered base pairing during replication such as methylmethane sulfonate (MMS), nitrosamine
• Anti-cancer drugs like Temozolomide or temodal alkylates guanine residues at positions 6 and 7 and interferes with DNA replication
• Other mutagen factors include Biotoxins (aflatoxin-B1) & Viruses (HPV)

Formation of Gene Mutations & Somatic Cells

• Gene mutations confined to a single gene are small DNA-sequence changes such as substitutions, small additions, and small deletions.
• Base substitutions replace the correct nucleotide by an incorrect one and are subdivided further
• Transitions are changes in which purine becomes purine or pyrimidine becomes pyrimidine
• Transversions are changes in which purine becomes pyrimidine and vice versa
• Frameshift mutations involve the addition or deletion of one or a few base pairs in protein coding regions
• The relative mutation frequency is the outcome of the race between repair and replication such that the more repair that takes place before replication, the lower the mutation frequency for a given amount of induced DNA damage
• Most so-called spontaneous mutations arise from replication of altered templates and are the result of oxidative damage or the deamination of 5-methyl cytosine to thymine transitions

Germ Cells

• The mechanism of production of gene mutations in germ cells is basically the same as that in somatic cells
• The majority of chemicals induce base substitutions, frameshifts, and small deletions through errors of DNA replication
• The spermatogonial stem cell is the major contributor to genetic risk assessment; it is generally present throughout the reproductive lifetime of an individual
• Each time spermatogonial stem cell divides, it produces a differentiating spermatogonium and a stem cell
• The stem cell can accumulate genetic damage from chronic exposures
• Oogenesis arrests the primary oocyte before birth and does no further S phase until the zygote which makes the oocyte resistant to induction of gene mutations by most chemicals

DNA Repair

• Two processes enable a cell to cope with extensive DNA damage
• The cell undergoes apoptosis is the damage is extensive
• If damage is less severe, repair processes return the DNA to its undamaged state through error-free repair or to an improved but still altered state through error-prone repair
• Most repair processes have basic principles such as damage recognition, removal of damage (except for strand breaks or cleavage of pyrimidine dimers), repair DNA synthesis, and ligation
• The major pathways by which DNA base damages are repaired involves removal of the damaged base
• The resulting gap can be filled by a DNA polymerase, followed by ligation to the parental DNA
• Oxidative damages, are important substrates for base excision repair
• Nucleotide excision repair (NER) provides a cell's ability to remove bulky lesions from DNA by means of damage recognition, incision, excision, repair synthesis, and ligation
• The DNA damage in actively transcribing genes, specifically the transcribed strand, is preferentially and thus more rapidly repaired than is the DNA damage in the rest of the genome
• NER protects the integrity of the transcription process
• Double-strand breaks trigger one or more DNA damage response systems to check cell cycle progression or induce apoptosis
• The two general pathways for repair of DNA double-strand breaks via homologous recombination and nonhomologous end joining
• DNA mismatch repair systems are important factors repair mismatched bases
• Principal steps of this system involves damage recognition by a specific protein that binds to the mismatch, stabilizing of the binding by the addition of one or more proteins, cutting of the DNA at a distance from the mismatch, excision past the mismatch & resynthesis & ligation

Testing Mutagenesis and the AMES Test

• This is a quick screening test for potential mutagenic compounds
• A strain of Salmonella with a defect in the histidine biosynthetic pathway is plated as a lawn on a medium containing minimal histidine (His)
• This will ensure cells remain alive but not enough to sustain proliferation
• Bacteria are spread on an agar plate with a small amount of histidine
• Small amounts of histidine in the growth medium allows bacteria to grow for an initial time and have to mutate
• When the histidine is depleted only bacteria that have mutated to gain the ability to produce its own histidine will survive
• The plate is incubated for 48 hours, and the mutagenicity of a substance is proportional to the number of colonies observed
• Rat liver extract can be added to simulate the effect of metabolism
• Compounds, like benzo[a]pyrene, are not mutagenic themselves but their metabolic products are
• The compound of interest is applied to a disc in the center of the plate and the plate is incubated overnight
• Different plates with increasing amounts of the compound are put up
• Sometimes liver extract is applied also to check for cellular conversions
• If the compound is mutagenic, it will cause a number of cells to revert such that so grow on the medium
• Non-mutagenic compounds will only show a few scattered colonies over the whole plate or spontaneous reversions
• A mutagenic compound has a linear dose response