Gene Mutation, DNA Repair, and Recombination

• The chapter discusses gene mutation, DNA repair, and homologous recombination.

Genetic Variation

• Genetic variation among individuals provides raw material for evolution.
• Genetic analysis relies on variants exhibiting phenotypic differences.
• Genetic variants arise through mutation and recombination.
• Mutation types include single nucleotide polymorphisms (SNPs), insertion-deletion polymorphisms (indels), copy number variations (CNVs), and chromosomal rearrangements (inversions, translocations).

Mutations vs Polymorphisms

• Mutations occur with a frequency of <1% in a population and typically lead to disease or disorder.
• Polymorphisms have a frequency >1% in a population and are usually neutral.
• A rare disease allele can become a polymorphism if it confers an advantage and increases in frequency.
• Heterozygosity for sickle cell anemia gives resistance to malaria
• Cystic fibrosis mutations in 5% of human population, give resistance to cholera and tuberculosis

Mutation Categories

• Spontaneous mutations occur naturally via normal biological or chemical processes.
• Induced mutations result from extraneous natural or artificial factors.
• Somatic mutations occur in non-germ cells and are non-heritable, only affecting direct descendants.
• Germ-line mutations, occurring in gametes, are inherited.

Point Mutations

• This involves changing one base pair into another in DNA.
• Localized mutations, also known as point mutations, occur at specific, identifiable positions within the genome.
• Consequences of point mutations depend on the type of sequence change and its location in the gene.

Base-pair Mutations

• Base-pair substitution mutations replace one nucleotide base pair with another.
• Transition mutations involve replacing one purine with another, or one pyrimidine with another which includes:
  • Pyrimidine replaces pyrimidine (T-C, U instead of T)
  • Purine replaces purine (A-G)
• Transversion mutations replace a pyrimidine with a purine or vice versa which includes:
  • A-T, G-T
  • A-C, G-C

Consequences of Point Mutations

• Synonymous mutations are base-pair changes not altering the amino acid due to genetic code redundancy, and are silent where only the genetic code changes without affecting the amino acid.
• Missense mutations are base-pair changes leading to an amino acid change such as a change in code and amino acid.
• Nonsense mutations are base-pair changes creating a stop codon, leading to premature translation termination and non-functional protein.

Missense Mutations

• This involves a change in code and amino acid.
• It affects a single nucleotide, resulting in a new triplet and altered amino acid within the protein.

Frameshift Mutations

• This arises from insertion or deletion of base pairs, leading to addition or deletion of mRNA nucleotides potentially altering the reading frame by either:
  • A premature STOP codon
  • A nonsensical amino acid sequence
  • An Excessively long sequence
• The wrong amino acid sequence is produced due to mutation; premature stop codons may also be produced.
• The mutation effect depends on where it occurs in the sequence
• The effect depends on if the mutation encodes STOP (UAA, UGA, or UAG)

Regulatory Mutations

• These mutations affect promoter, intron, and UTR regions, thereby only altering the amount of protein product.
• Mutation types are:
  • Promoter
  • Splicing
  • Cryptic Splicing

Promoter Mutation

• These alter consensus sequence nucleotides of promoters.
• Promoter interference disrupts transcription initiation.
• Promoter mutations cause mild to moderate transcription reductions, while abolishing transcription altogether.

Splicing Mutation

• Splicing mutations alter specific sequences at intron ends.
• Splicing errors result in mutant protein production due to intron sequence retention.

Cryptic Splice Sites

• Base-pair substitution mutations produce new splice sites.
• This process replaces or competes with normal mRNA processing splice sites.
• A base-pair substitution at position 110 in human β-globin intron 1 creates an A G cryptic splice site.
• The mutation also leaves 19 additional nucleotides in the mature mRNA.

Reversion Mutations

• Reversion mutations restore a wild-phenotype

Functional Effects of Mutation

• A wild-type phenotype occurs when an organism has 2 copies of the wild-type allele
• Mutant alleles can result in: - Loss-of-function - Gain-of-function

Dominant Negative Mutations

• Multimeric proteins are subject to dominant negative mutations.
• Mutations are dominant
• These cause a loss of function by preventing polypeptide interaction.
• These mutations prevent the protein from a whole resulting in a "spoiler" effect.

Gain-of-Function Mutations

• These are either hypermorphic or neomorphic.
• Hypermorphic mutations increase normal activity.
• Neomorphic mutations create novel activities not in the wild type form.
• These types of mutations are usually dominant

Mutations and Gene Products

• Loss-of-function mutations eliminates a gene product.
  • Null mutations: Tay-Sachs disease, double muscling
• Gain-of-function: new product or expression.
  • Huntington disease, Philadelphia chromosome
• Neutral mutations are mutations in noncoding regiosn.

How Replication Errors Arise

• Slippage during replication -> small insertions or deletions.
• Alterations in number of DNA repeats -> strand slippage.
• DNA polymerase of the replisome dissociates from the template
• The new replicated DNA forms a temporary hairpin.
• Resumption of replication -> replication of repeats and the number of repeats increases
• DNA polymerase occasionally inserts incorrect nucleotides generally due to mispairing and predominantly lead to point mutations.

Trinucleotide Repeat Expansion Disorders

• This Involves strand slippage mutations causing hereditary diseases which includes some hereditary diseases in humans.
• Variable number of DNA trinucleotide repeats are normally present in the wild-type alleles.
• Disorders are caused by increases in the number of repeats beyond a certain threshold such as: - Fragile X syndrome - Friedreich ataxia - Huntington disease - Jacobsen syndrome - Myotonic dystrophy (type I) - Spinal and bulbar muscular atrophy - Spinocerebellar ataxia (multiple forms)
• Length changes chromosome structure, which becomes methylated leading to transcriptional silencing

Nucleotide Mispairing

• Non-complementary base pairing called non-Watson-and-Crick base pairing
• This Includes G with T pairing or C with A pairing and is identified as an incorporated error.
• Replication of the incorporated error converts it into a mutation called replicated error

Spontaneous Nucleotide Base Changes

• Depurination occurs when a purine loss breaking the sugar's covalent bond.
• A lesion of this type is called an apurinic site, most are repaired before replication
• DNA Polymerase compensates normally; however an adenine is placed into the site during replication.
• Deamination occurs as a loss of an amino group (NH2)
• When a cytosine is deaminated -> oxygen atom -> uracil.
• DNA mismatch repair removes the uracil from the new DNA strand
• Creates the wild-type sequence with cytosine replacing uracil.
• When methylated cytosine is deaminated, a thymine base is produced
• The mismatch repair system restores the wild-type G-C pair.
• Repair failure will produce two sister chromatids a with the mutant A-T pair and one with the G-C pair

Base Damage

• DNA base damage from depurination and deamination is the most common cause of mutation
• (a) Unmethylated cytosine converts to uracil.
• (b) 5-methylcytosine deamination -> thymine (mismatched to guanine).
• (c) Mismatch repair creates a C G to T A transition or remove remove the thymine.

Mechanisms of Point-Mutation Induction

• Mutagens induce mutations and the different mutagens have different mutational specificities by type and site.
• Induced mutations are produced to examine what caused the damage, or repair responses to damage
• Mutagens interact with DNA in a way that leads to a specific sequence change

Chemical Mutagens

• Chemical mutagens:
  • Nucleotide base analogs
  • Deaminating Agents
  • Alkylating agents
  • Oxidizing Agents
  • Hydroxylating agents
  • Intercalating Agents

Mutation by incorporation of the Nucleotide Base Analog 5-Bromouridine (BU)

• A nucleotide base analog is a chemical that is a similar to a DNA Nucleotide base
• These pair normally and cannot be distinguished

Base Replacement

• Chemical compounds similar to nucleotides become incorporated in DNA
• This can cause mispairing for incorrect nucleotides to be incorporated during replication.

Intercalating Agents

• Some molecules can fit inbetween DNA base pairs.
• Some DNA intercalating agents distort the DNA Duplex
• This can result in DNA nicking/frameshift mutations

How UV generated photoproducts unite adjacent pyrimidines in DNA

• Photoproducts are aberrant structures caused by UV exposure that have additional bonds involving nucleotides.
• Thymine dimer is a photoproduct formed between 5 and 6 carbons of adjacent thymines.
• 6-4 photoproduct is formed by covalent linking of the 6th and 4th carbon on one thymine and carbon of another.

Consequences of Photoproducts

• DNA repair systems normally fix photoproducts
• Those not repaired cause replication disruption.
• Disruptions lead to mutations/skin cancer**

What was found from Gene mutation

• The result of single nucleotide differences in IGF1 gene cause diversity in breed size.
• Also that a missense mutation in Aggrecan resulted in genetic variations

Direct Repair of DNA Damage

• Direct repair to identify and reverse the DNA
• One direct repair is the proofreading of polymerase activity
• Other repair systems also carry out the DNA's repair

Common Systems

• Photoreactive repair: induced by UV via photolyase activating visible light.
• Base excision: incorrected repaired by new strand (nick translation)
• Nucleotide excision repair segment from replacement synthesis
• Mismatch segment following segment synthesis

Photoactivation repair

• Thymine dimers are removed and caused by UV light
• Repair is facilitated by the photoreactivation enzyme
• Bacteria, eukaryotes, and plants undergo repair
• Requires energy from light to break the bonds.
• This process Is not necessarily essential for survival

Base Excision Repair (BER)

BER fixes a base or replace a base (multistep).
DNA glycosylases recognize bases + produce A P site with nick location.
Ligation -> sugar phosphate backbone bonds

Nucleotide Excision Repair (NER)

• This repairs bulky lesions which involves uvr genes
• Ultraviolet (UV) repair is often used

Homologous Recombination Repair

• Uses sister chromatids for DMA polymerases to copy
• Double strand breaks can be repaired as synthesis dependent
• SDSA happens by trimming of strands
• Rad51 occurs for integrity with the duplex formation/D loop forms to make synthesis/template.