Mutations and DNA Repair

What are mutations?

Heritable change in nucleotide sequence of DNA

Somatic vs. Germline Mutations

Somatic mutations in somatic cells → inherited by daughter cells but not progeny

Germ-line mutations in gametes and transmitted to progeny

Where can mutations occur? What is the consequence?

Anywhere in the genome → can have no functional consequences or affect gene expression

Mutations in protein coding region

Can affect amino acid sequence
Substitution - missense, nonsense, silent
Indels (insertion/deletion) - frameshift or in frame

Mutations in noncoding regions

Can affect rate of transcription or post-transcriptional processes
Promoter/enhancer mutations
Splicing mutations
Mutations in 5’ and 3’ UTR

Types of gene mutations

Base substitution, insertions/deletions, expanding nucleotide repeats

Base Substitution

Alteration of a single nucleotide (point mutation)

Transition vs. Transversion

Transition: pyrimidine → pyrimidine OR purine → purine
Transversion: purine → pyrimidine OR pyrimidine → purine

Missense mutation (nonsynonymous mutation)

Amino acid is changed

Nonsense mutation

Codon changed to stop codon

Silent mutation (synonymous mutation)

Amino acid is NOT changed
Occurs at degenerate codons

Conservative Mutation

Chemical properties of mutant amino acid are similar to the original amino acid (same R group)
Often has NO effect on the function of the protein

Nonconservative mutation

Chemical properties of mutant amino acid are different from original amino acid (Different R group)

Often has an effect on the protein

Frameshift mutation (in coding regions)

Insertions or deletions that are NOT multiples of 3 alters reading frame
C-terminal portion will be mutant amino acid sequence

In-frame mutation (in coding regions)

Insertions or deletions in multiples of 3 does not alter reading frame

C-terminal portion will have wild-type amino acid sequence

Expanding nucleotide repeats (triplet/trinucleotide repeat disorders)

CNG repeats multiple times than what is originally in the amino acid sequence

Shows anticipation → becomes more severe and earlier each generation
Ex. Fragile X and Huntington Disease

What causes nucleotide repeat expansions?

Strand slippage during replication
Hairpin forms on newly synthesized DNA strand
Part of template is replicated twice

Daughter cells contain DNA and is now the template strand

Loss-of-function (LOF) mutations

Reduced or abolished activity of the gene product

Usually recessive inheritance
Null (amorphic) - block function of gene product

Hypomorphic - gene product has weak activity

Gain-of-function (GOF) mutations

Enhance an activity or confer a new activity or location of an activity

Usually dominant inheritance

Hypermorphic - generate more gene product

Neomorphic - generate gene product with new function or at inappropriate time/place

Forward mutation

Wild type allele changed to different allele

A+ → A-

Reverse mutation (revertant)

Mutant allele changes back to wild type

A- → A+

Frequency reversion is less than frequency forward mutations (depends on type)
Point mutation > Indel

Suppressor mutation hides effect of another mutation

True revertant: original mutation changed back to wild-type allele

Two kinds of suppressor mutations

Intergenic Suppressor - second mutation in DIFFERENT gene restores gene function; has two mutated genes; also called second site suppressor

Intragenic Suppressor - second mutation in the SAME gene restores function; one gene has two different mutations

Three ways for Intragenic Suppression to occur

1. Suppressor mutation in same codon as first mutation, but same amino acid is encoded

2. Suppressor mutation restores reading frame of frame shift mutation

3. Suppressor mutation is 2nd missense substitution that restores protein function

Spontaneous mutations

Arise without exposure to any external agents

Induced mutations

Produced by interactions between DNA and a chemical or physical mutagen

Examples of spontaneous mutations

Mistakes in DNA replication (Base sub)

Depurination/Deamination (Base sub)

Strand slippage (Indel)

Unequal crossing over (Indel)

Examples of induced mutations

Ultraviolet light (pyrimidine dimers)

Ionizing radiation (double strand breaks)

Depurination

Loss of purine due to spontaneous breakage of glycosidic bond of a nucleotide

Creates apurinic (AP) site in DNA strand

During replication, DNA polymerase puts adenine opposite AP site

(Keeps replicating A bc AP site is new template strand)

Spontaneous deamination of 5-methylcytosine

Deamination - loss of an amino group form a base

5meC gets deaminated → Results in C to T transitions

Not recognized by any repair mechanism → mutations frequent

Two mechanisms for formation of indels

Strand slippage & Unequal crossing over

DNA Damage caused by UV light

Abnormal covalent bonds between adjacent pyrimidines (thymine dimers)

Distorts double helix

DNA replication stalls bc complementary adenines of new strand can’t form H-bonds with thymine dimers

DNA Damage caused by ionizing radiation

Double-strand breaks

Unrepaired breaks block DNA replication or chromosome abnormalities → cell death or cancer

Mismatch repair

Enzymes cut out a section of the newly synthesized strand of DNA and replace it with new nucleotides

Repair errors during or immediately following replication

Base excision repair (BER)

Glycosylase enzymes recognize and remove specific types of modified bases

Entire nucleotide is removed and a section of polynucleotide strand is replaced

Nucleotide excision repair (NER)

Removes and replaces many types of damaged DNA that distort DNA structure

Two strands of DNA are separated, a section of the DNA containing the distortion is removed, DNA polymerase fills in the gap, and DNA ligase seals the gap

Homologous recombination (HR)

Similar mechanism during meiosis

Repair accomplished using sequence from homologous chromosome or sister chromatid

Typically error-free

Nonhomologus end joining (NHEJ)

Before DNA replication (G1)

Error-prone

Ku binding → Ligation