Mutations and DNA Repair

What are mutations? What are two locations where mutations can occur in?

Heritable changes in nucleotide sequences, and they can occur in the gene and the chromosome

Do all mutations result in phenotypic consequences?

No, it depends on the gene and location

Compare gene and chromosome mutations on a large scale?

Gene: small alterations that affect a single gene or locus

Chromosome: large-scale that affects chromosome structure (ex: inversion) or chromosome number (aneuploidy)

Compare somatic versus germline mutations (location, what other cells it affects)

Somatic mutations occur in somatic cells (all other cells besides gametes), and germ-line mutations occur in gametes or gamete precursors (germline lineage or germ cells)

For somatic, the altered sequence is inherited by daughter cells (the cells produced during mitosis) but not offspring. However, it is the opposite for germ-line cells. The altered sequence is inherited by offspring and can be transmitted to the next generation

Why are germ-line mutations more detrimental than somatic mutations?

These mutations affect more than just the primary organism; germ-line mutations persist to offspring. Additionally, the mutation does have a “bigger” effect. By “bigger,” a mutation in the germ-line cells affects the basis DNA for all cells in the offspring (and can be passed down to other offspring). Therefore, all cells of the offspring will carry the mutation (not always and will not always have the mutation be active). Somatic mutations still have an impact, but they only target a specific kind of cell (ex: progeny of affected cell/gene)

True or false: mutations only occur in coding regions and always result in a consequence

False-they can occur in coding and noncoding regions, and they do not have to have a functional consequence/affect the expression of the gene

Coding vs noncoding regions mutations (what do they affect and what generally happens)

Coding:

• can affect amino acid sequence (commonly in mRNA before being transcribed)

• ex: substituions (nonsense, silent, or missense) or indels (insertion/deletion) or frameshift or in-frame

Noncoding:

• can affect rate of transcription or post-transcriptional processes

• ex: promoter/enhancer mutations to affect rate or transcription; splicing mutations can remove 5’ or 3’ splicing sites to crew new splice sites; mutations in 5—UTR and 3’-UTR can influence mRNA stability and translation initiation

What are the three different types of gene mutations?

1. Base substitution

2. Insertions and deletions

3. expanding nucleotide repeats

What is a base substitution and what are the different types

A single nucleotide is altered (point mutation. There are two types:

Transition: pyrimidine to pyrimidine (ex: C to T) OR purine to purine (adenine to guanine)

Transversion: purine to pyrimidine (A to C) or pyrimidine to purine (T to G)

How many possible transition mutations are there? What about transversions

Four possible transitions

Eight possible transversions

What are three possible outcomes/consequences on base substitutions in protein coding sequences?

1. Missense mutation: an amino acid is changed (nonsynonymous mutation)

2. Nonsense: codon is change to stop codon=function of C-terminal amino acid sequences

3. Silent mutationL amino acid is not changed (synonymous mutation) because it occurs at degenerate codon (aka the third spot where it can be changed but not affect the identity of the amino acid)

Of the missense mutations, what are the two kinds

1. Conservative mutation: chemical properties of mutant amino acid is similar to the original one because they have the same R (at least same kind) functional group. Therefore, they have no effect on the function of the protein

2. (2) nonconservative: chemical properties of mutant are different because their is a different functional group; it does not always (but often) it it can have an effect on the function of the protein

How long are insertions or deletions?

Can be a few nucleotides or 1000s of nucleotides

What are the two possible outcomes/consequences on insertions/deletions (indels)? Describe them

1. Frameshift mutation: occurs in coding regions, and it alters the translation reading frame; result of an indel that is not a multiple of 3 (because bases are read in groups of three=codons), so the C-terminal portion of the polypeptide will be affected

2. In-frame mutation: also occurs in coding regions, but the indel is a multiple of 3. therefore, the reading frame is not affected, but there will be some additional or missing amino acids (still not as bad as shifting the entire sequence like with frameshift mutations). the C-terminal portion will have a normal (wild-type) amino acid sequence

What portion of the polypeptide chain do we look at to see whether or not we have a frameshift or in-frame mutation as result of an indel?

The C-terminal. If it shows a mutant phenotype, we can infer a frameshift mutation has occurred (not in a multiple of three), but if the C-terminal depicts a wild type phenotype, then we can infer an in-frame mutation has occurred

What nucleotides are involved in expanding nucleotide repeats?

Usually CNG

N=any nucleotide

What are some effects of expanding nucleotide repeats? (describe them, what they are called, and some characteristics)

Genetic disorders, can be seen in humans, and they are typically inherited (because they occur in germline cells). They are called triplet (or trinucleotide) repeat disorders. They show anticipation (aka disease becomes more severe with each generation), and the number of copies can correlate to the severity and/or age of onset of the disorder

Do expanding nucleotide repeats occur in any specific region (aka are they exclusive to coding or noncoding)?

They can occur in noncoding or coding regions

How is Huntington disease an example of triplet repeat disorder?

It is caused by CAG repeats.

Even if an individual has some expanding nucleotide repeats, will they necessarily have a disease/phenotypic effect?

No. For Huntington’s disease, individuals can have CAG repeats and be unaffected. Specifically, if an individual has 9-37 repeats, they are usually unaffected. However, disease-causing alleles have 37-121 repeats

What is the cause of nucleotide repeat expansions?

Strand slippage; a hairpin can form on a newly synthesized strand, exposing a section of the template strand that was just replicated. Therefore, this section will be synthesized twice, leading to repeats

How do nucleotide repeat expansion differ from insertions?

Nucleotide repeat expansion is caused by slippage, and these sequences are always repeats. Insertions do not have to be repeats of a sequence; they are just an inserted sequence. Also, while they can be caused by slippage, they can also be caused by unequal crossing over

What kind of blotting technique can we use to see a genetic disorder in humans?

Southern blot

What is a loss-of-function (LOF) mutation?

The gene activity/product will be reduced or abolished

What kind of inheritance do loss-of-function mutations have?

Recessive inheritance

What are two categories of loss-of-function mutations? Describe them

Null (amorphic) mutations: complete block function of gene product (ex: deleting entire gene) (abolished)

Hypomorphic mutations: gene product/activity has been weakened; it is still detectable (reduced)

What is a gain-of-function mutation?

Where the effect enhances an activity/product or results ina. new activity or location of an activity

What kind of inheritance does a gain-of-function mutation have?

Dominant inheritance

What are the two categories of gain-of-function mutations?

Hypermorphic mutations: generating more gene product or the same amount of an EFFICIENT gene product (works better but does not necessarily mean it is good) (not completely random/altered)

Neomorphic mutations: results in a gene product with a new function or that is expressed at inappropriate time and place (completely alteration in some way

LOF and GOF mutations can occur in the same gene, but do they differ in effects?

Yes, take a look at this example (the mutations occur in different spots on the gene)

Loss of function mutation in the sonic hedgehog mutation of humans results in holoprosencephaly (autosomal dominant inheritance and results in a facial phenotype caused by haploinsufficiency because one functional copy is not enough)

Gain of function mutation in enhance of the same gene results in polydactyly (specifically neomorphic→the gene will be transcribed in an abnormal spot; autosomal dominant inheritance)

What is a forward mutation? What about a reverse mutation

Wild type allele to a different allele (ex: A+ = red eyes but changed to A- which is white eyes)

A reverse mutation is when the mutant allele changes back to wild type allele (A- to A+).

How do forward and reverse mutations differ in frequency?

The reverse mutation occurs less frequently than forward mutations; reverse mutation frequency depends on the type of mutation (ex: it is more common to see a reverse mutation in a point substitution than a indel because it is easier to reverse one base than remove/add sequences)

What is a suppressor mutation? (and how does it differ from a revertant)

When a mutation hides the effect of another mutations. They appear to be revertants, but are actually caused by a different mechanism. Specifically, if an organism has a suppressor mutation, they have two phenotypes (1) mutant (2) supperepressor

To summarize, List of all the phenotypic mutations we have covered

-LOF and GOF

-Forward and Reverse

-Suppressor (intergenic and intragenic suppressor)

What is a true revertant?

when an original mutation is changed back to wild-type allele (one mutation not two mutations like suppressor)

What are the two kind of suppressor mutations? Describe them

Intergenic suppressor: second mutation in a DIFFERENT gene restores gene function—this is known as a second site suppressor (there are now two mutated genes)

Intragenic suppressor: second mutation that restores gene function is in the SAME gene (one gene but two different mutations)

What is a second site suppressor

Intergenic suppressor; the site of where the second mutation is on the DIFFERENT gene

Explain how intergeneic suppressor works by using Gene A and B that produce polypeptides that interact to be functional

If either Gene A or Gene B has a mutant phenotype (not both), there will be no function. However, if both are mutated, there will be a function (aka the second mutation counterbalances the first mutation)

What are the three ways intragenic (reminder same gene) suppression occurs?

1. Suppressor mutation is in the SAME CODON as the first mutation, but the same amino acid is encoded; different base on the same codon is changed to restore coding back to original codon (aka if a mutation change a UCU codon to ACU, the amino acid goes from being serine to heronine. however, if a second mutation changes ACU to AGU, the codon now codes the amino acid back to serine; this is two different mutations—not fixing the first one)

2. The mutation restores the reading frame of a frameshift mutation

3. The mutation is a second missense substitution (2nd mutation at a different codon that will produce a different amino acid) to restore the conformation of the protein so the function is restored (aka the new amino acid helps restore the function)

How do mutations arise? (2)

Either spontaneously (without expore to any agents) or they are induced (due to interactions between DNA and a chemical (includes nucleotides)/physical mutagen—any environmental agent that really increase the rate of mutation above the spontaneous rate)

Source of mutation:Spontaneous or Induced? Type of mutation? Mistakes in DNA replicaiton

Spontaneous and base substitution

Source of mutation:Spontaneous or Induced? Type of mutation? Depurination and deamination

Spontaneous and base substitution

Source of mutation:Spontaneous or Induced? Type of mutation? Strand slippage during DNA replication

Spontaneous and indels

Source of mutation:Spontaneous or Induced? Type of mutation?: Unequal crossing over

Spontaneous and indels

Source of mutation:Spontaneous or Induced? Type of mutation? Ultraviolet light

Induced and pyrimidine dimers

Source of mutation:Spontaneous or Induced? Type of mutation? Ionizing radiation

Induced and double strand breaks

What happens during depurination? What was the cause? What is the effect?

Depurination involves a loss of a purine=base substitution (adenine and guanine)

Cause: spontaneous breakage of the glycosidic bond of a nucleotide (links base to backbone)

Effect: This creates an apurininc (AP) site in DNA, but during replication, DNA polymerase will put an adenine opposite of this site, resulting in a transition mutation (if the original one was a G)

What happens during deamination? What is the effect?

An amino group is lose from a base, and 5-meC is commonly deaminated

Effect: many more mutations occur after the deamination of 5-meC. Specifically, there can be cytosine to thymine transitions; this is not recognized by any repair mechanism

What is the epigenetic mark of deamination?

5meC

What are the two spontaneous causes of the formation of indels?

Strand slippage: similar to what happens in expanding nucleotide repeats, where a section slips/forms a hairpin for its complementary section of the DNA to get repeated again. however, these do not involve as big of a hairpin—usually only one base slips

Unequal crossing over: misalignment of chromosomes

What is the effect of exposure to ultraviolet (UV) radiation (aka the effect of this induced mutation)

UV light creates abnormal covalent bonds between adjacent pyrimidines (C, T, and U), known as thymine dimers. This results in a distortion in the double helix, stalling DNA replication because the distortion prevents the necessary enzymes from binding to the DNA and completementary adenine cannot form hydrogen bonds with the thymine dimer

What is the effect of exposure to ionizing radiation?

Double-strand breaks caused by ionizing radiation, where DNA replication errors and unintended cleavage by nuclear enzymes occur. Any unrepaired breaks can inhibit DNA replication and result in chromosome abnormalities (cell death or cancer)

What happens during mismatch repair?

Mismatched bases and other DNA lesions (damaged section) and correct it by a mismatched par: (1) having an enyme cut out the section of the just synthesized DNA and will (2) replace it with few nucleotides. This will be used during or immediately following replication

Why do we need pathways for replication?

Becsaue cells are not ALWAYS replicating, so we need mechanisms to that work outside of replication

What happens during Base exclusion repair (BER)

Gycosylase enzymes recognize and remove modifed breaks. and then, the entire nucleotide is removed, and the section of the polynucleotide sequence will be replaced

*mainly occurs with small.small errors

What happens during nucleotide excision repair (NER)?

It will remove and replace many types of damaged DNA that do distort the structure and alter the function (more specific than BER). Two strands of DNA are separated, with one having the distortion, will have it remove and replace via DNA polymerase. DNA ligase will come in to sea the filled in gap

What are two ways to repair a double-strand break (often caused by ionizing radiation)?

1. Homologous recombination (HR): repair is accomplished through a sequence of a homologous chromosome or sister chromatid; error-free; similar mechanism as during meiosis

2. Nonhomologous end joining (NHEJ): occurs before DNA replication; no sister chromatid is present ( it is in G1 aka the only phase before replication really); error prone (pasting back together without a template to repair what was lost. therefore, overall, some DNA is lost) (think of Ku binding)

*CRISPR relies on these for genome editing

To summarize, what mutation repair mechanism have we discussed

1. Mismatch repair (mismatch pair)

2. Base exclusion repair (BER) (entire nucleotide is removed ultimately)

3. Nucleotide expression repair (NER): damaged DNA that distorts the structure is removed

4. Homologous recombination (HR): (double-stranded break resolution): sister chromatid + error-free

5. Nonhomologous end joining (NHEJ): G1 and no sister chromatid + error prone

To summarize, what are some causes of mutations we discussed (spontaneous vs induced)?

Spontaneous: depurination and demamination (base substituion) slippage and unequal crossing over (indels)

Induced: Uv radiation (thymine dimer) and ionizing radiation (double strand breaks)