Understanding DNA Mutations

• Change in DNA sequence is defined as a mutation.
• Every organism carries mutant alleles that may or may not be expressed.
• New mutations can arise each generation.
• Mutations within individual genes involve a change in nucleotide sequence, impacting protein structure or gene expression.

Causes of Mutation

• DNA replication errors.
• Spontaneous mutations.
• Exposure to chemicals and irradiation like ultraviolet light.

Somatic vs. Germ-Line Mutations

• Somatic mutations: Occur in body cells, not inherited. Can potentially lead to cancer.
• Germ-line mutations: Occur in gametes (egg and sperm), are inherited by offspring.

Base-Pair Substitution Mutations

• This involves the replacement of one nucleotide base pair with another.
• Silent mutation: A base-pair change does not alter the amino acid due to genetic code redundancy.
• Missense mutation: A base-pair change results in an amino acid change in the protein.
• Nonsense mutation: A base-pair change creates a stop codon, prematurely terminating protein synthesis.

Types of Base Substitution.

• Changes one codon for an amino acid to another codon for the same amino acid.
• There is no change in the amino acid (silent mutation).
• Changes one codon for one amino acid to codon for another amino acid.
• Effect of changing an amino acid depends on its role in protein function.

Severity of Missense Mutations.

• Missense mutations: can widely vary in their severity.
• Conservative amino acid substitution involves substituting a chemically similar amino acid, less likely to alter protein function; often called a neutral mutation.
• Nonconservative amino acid substitution substitutes a chemically different amino acid, more likely to alter protein function.
• Consequences for protein depend on the function of the amino acid within the 3D structure.

Key Points about Genetic Code and Mutations.

• Most amino acids correspond to more than one codons.
• Mutation in the first or second nucleotide of the three letter codon is more likely to result in an amino acid change than a mutation in the third nucleotide.

Types of Base Substitution Mutation: Nonsense.

• Involves changing a codon for an amino acid into a translation termination (stop) codon.
• Nonsense mutation results in premature termination of translation
• In general truncated polypeptides created due to nonsense mutation are non functional.

Frameshift Mutation

• This involves the addition or deletion of one or more bases, altering the reading frame
• Alters all downstream amino acids from the point of mutation.

Regulatory Mutations

• Some point mutations alter the amount (but not the amino acid sequence) of protein product produced by a gene.
• Affect regions such as promoters and other regulatory protein binding sites.
• These that affect regulatory proteins such as the transcription and regulatory proteins or promoter mutations may alter transcription levels.

Origin of Mutation

• Spontaneous mutation: Arise in absence of known mutagens, may be caused by errors in DNA replication, providing a "background rate" of mutation.
• Induced mutation: Is an action by a mutagen, an environmental agent that alters nucleotide sequence. The process of inducing mutations by mutagens is called mutagenesis.

Types of Spontaneous Mutation

• DNA replication errors: DNA polymerase can insert the wrong nucleotide, but it is usually corrected by the 3’ to 5’ 'proofreading' exonuclease action of the DNA polymerase 99% of the time.

Trinucleotide Repeat Disorders

• Caused by DNA polymerase slipping during DNA replication and increasing the number of tri nucleotide repeats within a gene
• Results in longer stretches of the same amino acid within the protein.
• Huntington's disease is from increase in length of a polyglutamine region in the Huntingtin protein. Normal is 28 CAG repeats, disease symptoms are 36 or more CAG repeats.

Spontaneous Nucleotide Base Changes

• DNA nucleotide bases can convert to alternative structures called tautomers.
• Alternative structures create slight differences in bonding and placement of hydrogens that lead to Tautomeric shifts, that can also lead to base-pair mismatches and incorporation of incorrect bases during replication.
• An example of this is an amino to imino shift or a keto to enol shift.
• This is a common form of DNA lesion.

Transposable Elements

• Have the ability to cause mutations on insertion into genes, rare in eukaryotes.
• These are found integrated in the genomes of all prokaryotes and eukaryotes (including humans).
• Retrotransposons found in eukaryotes (move position via RNA intermediate and reverse transcriptase).
• DNA transposons found in both prokaryote and eukaryote genomes; use protein transposase to move position.
• All can cause mutations by inserting into a gene.

DNA Transposons

• Are called Insertion Sequence(IS) elements in bacteria E. coli.
• Each IS element contains about 1000 bp of DNA.
• Carry a transposase gene bracketed by a short, inverted repeat (IR) sequence.
• Transposase binds to each inverted repeat (IR) and blunt end cuts on both sides of the IS element.
• Inserts into a gene will create an insertion mutation that blocks translation of a protein.
• Transposase cuts the new target site in the genome with staggered cuts (not target sequence specific).

Types of Induced Mutations.

• Induced mutations arise from interactions between DNA and physical, chemical, or biological agents.
• Nucleotide base analogs: chemical compounds similar to normal bases, but with altered pairing properties.
• Deaminating agents: remove NH2 groups from bases.
• Alkylating agents: add methyl (-CH3) or ethyl group (-CH2-CH3) to bases.
• Hydroxylating agents: add hydroxyl group (-OH) to bases.
• Oxidative agents: addition of oxygen or removal of hydrogen.
• Intercalating agents: Insert themselves between base pairs, distorting DNA.
• Radiation induced DNA damage: ultraviolet light

Induced Mutations: Base Analogs.

• Base analogs are similar to nitrogenous bases of DNA, but have altered pairing properties
• E.g. 5-bromouracil (5-BU) replaces thymine and causes an AT -> GC transition.

Induced Mutations: Alkylating Agents

• Alkylating agents modify base structure by adding methyl or ethyl groups, which can result in altered base pairing
• E.g. EMS (ethyl methanesulfonate)

Induced Mutations: Intercalating Agents

• Intercalating agents are flat, planar molecules that can intercalate between base pairs that causes a distortion in the DNA helix which disrupts DNA replication that causes addition or deletion of nucleotides leading to frameshift mutations.
• E.g. proflavin, acridine orange, ethidium bromide.

DNA Repair Mechanisms

• Organisms employ several mechanisms to repair DNA damage.
• Photoreactivation repair: DNA damage repaired by photoreactivation enzyme (PRE) cleaves the thymine dimer bond. This mechanism is not present in humans. Thymine dimers are commonly caused by ultraviolet light
• Base excision repair: Performed by a base specific DNA glycosylase (uracil DNA glycosylase), AP endonuclease, DNA polymerase and DNA ligase, in eukaryotes. Excision is recognised as a noncomplementary base pair and replaced with the complementary base.
• Nucleotide excision repair: The uvrA, uvrB and uvrC proteins recognise the lesion and excises 13 nucleotides. DNA polymerase I then synthesises the new DNA strand and DNA ligase joins the single stranded nick.
• Human diseases can be caused by mutations in genes that encode proteins involved in DNA repair mechanisms that includes Xeroderma pigmentosum.

Human Diseases from Mutation of DNA Repair Genes

• Mutations in human genes whose proteins are involved in DNA repair mechanisms can cause hereditary cancer.
• Somatic mutations in human genes whose proteins are involved in DNA repair mechanisms can also lead to cancer.
• BRCA2, MSH2, MLH1 genes are all involved in hereditary cancer.
• Xeroderma pigmentosum disease symptoms are caused by mutant alleles of nucleotide excision repair mechanism.