Ch 13 Gene Mutations, Transposable Elements and DNA Repair

Dominant Negative mutation

(antimorph) − Individuals heterozygous for the mutation phenocopy nulls − Often affect proteins that homodimerize, one mutant copy in the dimer impairs function of the wild-type copy − Dominant

Chromosomal rearrangement mutagens

- Deletions, duplications, inversions, translocations - Formaldehyde, X-rays, homologous recombination between repeated elements, e.g. transposons

• Indel mutagens

- Insertion/deletion of single base pairs - Ethidium bromide, proflavin, quinacrine mustards, acridine orange

• Hereditary colon cancer (HNPCC)

- Mismatch Repair defective (MutL homolog)

induced mutation causes

Exposure to chemical mutagen or source of radiation (gamma rays, x-rays, plutonium...)

Mutations can reveal insights into fundamental biological processes [Drosophila]

Mutations in the Drosophila eyeless gene revealed that eyeless encodes a master regulator of eye development in flies Drosophila eyeless mutants do not make eyes Ectopic eyeless expression eyes develop in new places Thus, eyeless is both necessary and sufficient for induction of eye development in Drosophila

Point mutation

single base substitution

Normal Base Pairing in DNA

• Purines - Adenine (A) - Guanine (G) • Pyrimidines - Thymine (T) - Cytosine (C) A:T G:C

• Xeroderma pigmentosum

(UV sensitivity; skin cancer): - Nucleotide Excision Repair defective

Ionizing radiation

(X-rays, gamma rays, cosmic rays) - Often make double-stranded breaks in DNA molecule (break phosphodiester bonds on both strands!) - Create free radicals which can alter the structure of DNA bases - Repair can result in chromosome rearrangements *Chromosomal Rearrangement Mutagen*

Gain-of-Function mutation

(hypermorph) − Increases activity of a gene − May result from overexpression, constitutive (unregulated) expression, or constitutive activity (lacIq c=t at -35 of lacI promoter = 10 fold more Lac repressor!) − Usually dominant

Loss-of-Function mutation

(hypomorph = partial loss) − Partially or completely impairs the function of a gene product − May result from decreased expression or stability, improper folding, disrupted protein-protein interactions, etc. − Usually recessive (unless haploinsufficient)

Deamination

A DNA base loses an amino group (can occur spontaneously or be induced) Deamination of 5-methylcytosine to form thymine and from normal cytosine to uracil Important: > Many cytosine bases in the human genome are methylated in CpG islands > Repair machinery recognizes Uracil as base error in DNA, but not Thymine

Ataxia Telangiectasia (ATM)

Defects in muscle coordination, high cancer susceptibility - DNA damage fails to trigger cell cycle arrest to give time for repair

the Ames test

How to measure the mutagenicity of a chemical: the Ames test • most agents that cause cancer (carcinogens) are also mutagens, so measuring mutagenicity provides an initial screen for potentially hazardous agents • the Ames test is a genetic assay for mutations in bacteria for detection of chemical mutagens • histidine-requiring (His- ) mutants of the bacterium Salmonella typhimurium, containing base substitution or frameshift mutation, are tested for backmutation to His+ • bacterial strains made more sensitive to mutagenesis by incorporation of mutant alleles: - inactivate excision-repair system - make cells more permeable to external molecules

Spontaneous mutation causes

Replication errors, background irradiation, chemical reaction with bases in DNA

Insertional mutagens

Transposable elements (natural and engineered)

Depurination is a spontaneous chemical change in which

a DNA molecule loses a purine base During the next round of DNA replication, the new DNA strand could have any base incorporated (A most often).

Insertions

addition of 1 or more basepairs of DNA

Alkylation

addition of alkyl chain to base induced by EMS

Essential genes can only be inactivated as

conditional mutants (or mutants must be maintained as heterozygotes)

Indel

either an insertion or a deletion, or a complex mutation that has both a deletion and insertion

A somatic mutation during embryogenesis yields an organism that is

genotypically a mixture (mosaic) of normal/mutant tissue

why does trinucleotide repeat expansion occur ?

replication slippage

Intercalating agents

wedge themselves between consecutive bases in a DNA molecule • Intercalation distorts the shape of the helix • Distortion leads to single bp insertions and deletions upon replication exs. proflavin and acridine orange INDEL MUTAGEN

Base excision repair (BER)

when single base is altered (subtle damage) remove base, then remove sugar • Deamination of cytosine = uracil, uracil removed by DNA uracil glycosylase. = an apyrimidinic site • The BER system: 1. Removes the base 2. Removes the sugar and phosphate 3. Repairs the gap 4. Ligates the backbone

UV Radiation

*Chromosomal Rearrangement Mutagen* - Causes adjacent pyrimidine bases on one DNA strand to bond and form pyrimidine dimers - Can also generate free radicals and cause chromosome rearrangements

Mismatched bases are standard bases. How does repair system determine what partner to eliminate?

- Old strand methylated, new strand methylation lags behind moving replication fork - Mismatch repair system preferentially excises nucleotides from the non-methylated daughter strand

Example of a mutation hotspot:

Trinucleotide repeats

Frameshift mutations

change the reading frame of the codons in the mRNA (insertion or deletion that is not a multiple of three nucleotides)

reverse mutation

changes a mutant phenotype back to the wildtype phenotype

Point mutations in protein-coding regions:

• missense (e.g., nonsynonymous) substitutions result in one amino acid being replaced with another - may or may not change protein function • silent (e.g., synonymous) substitutions in DNA do not change the amino acid sequence - genetic code is redundant - usually do not change protein function, but can if change is from a codon with a highly abundant tRNA to a rare isoaccepting tRNA or vice versa • nonsense mutations create new stop codons - 3 types: amber, ochre, and opal for 3 stop codons

Neomorph

− Gene product gains new function − May result from expression in wrong tissue or at wrong time, or by altered protein structure − Usually dominant

Null mutation

(amorph) − A complete loss-of-function mutation − May result from complete gene deletion, premature stop codon, or indel that changes reading frame (frameshift) − Could also be a missense mutation − Usually recessive (unless haploinsufficient)

Base-substitution mutagens

- Base analogs - chemicals with structure similar to DNA base - Alkylating agents - chemicals that modify DNA base Base analog: 5‐Bromouracil (Bu); DNA polymerase can't tell the difference between thymine & the analog Base analog: mispairing mutagenesis by 5-bromouracil a-Bu base pair= normal basepairing G- bu base pair= changed basepairing

Intergenic suppressor examples

- Mutation that disrupts protein-protein interaction suppressed by compensatory mutation in binding partner - Internal stop codon suppressed by suppressor tRNA (mutant tRNA with anticodon that matches stop codon) A suppressor mutation is a second mutation that alleviates or reverts the phenotypic effects of an already existing mutation in a process defined synthetic rescue. Genetic suppression therefore restores the phenotype seen prior to the original background mutation.

DNA Repair Mechanisms

1. Mismatch repair fixes incorrectly matched base pairs 2. The AP endonuclease system repairs nucleotide sites at which the base has been lost or removed 3. Special enzymes repair damage caused to DNA by ultraviolet light (e.g., photoreactivation repair) 4. Excision repair works on a wide variety of damaged DNA

For any DNA base, there is ____ possible transition and --- possible transversions

1; 2 Yet... Transitions are observed more often than transversions.

Intragenic suppressor example

2 bp deletion compensated with 2 bp insertion

how many DNA alterations become a mutation?

<1/1,000

mutational hotspots

Certain DNA sequences are called mutational hotspots because they are more likely to undergo mutation than others For example, sites of cytosine methylation are usually highly mutable (C in CpG islands in humans frequently methylated)

Mutagens result in permanent genome changes

Somatic = cancer, germline = birth defects, evolution

photoreactivation repair

Special enzymes repair damage caused to DNA by ultraviolet light

hydroxylation

addition of hydroxyl group to a base, caused by hydroxylamine

insertion

addition of one or more nucleotides

Somatic mutations

affect individual Germline mutations are inherited; somatic mutations are not

Germline mutations

affect progeny Germline mutations are inherited; somatic mutations are not

Transposable elements

are DNA sequences that can "jump" from one position to another or from one DNA molecule to another. • Flanked by direct repeats & inverted repeats. • Direct repeats mark the site of insertion into target DNA - sequences vary • Inverted repeats are recognized by transposase

Transversion

base substitution in which a pur replaces a pyr or pyr replaces pur

Transition

base substitution in which a purine replaces a purine or pyr replaces a pyr

disease mutation cancer

cancer cells often have defects in the mechanisms that sense and repair DNA damage, which increases accumulation of mutations

Endogenous factors

cause of mutation (spontaneous mutations) - DNA polymerase error - Strand slippage during replication - Reactive oxides produced by metabolism - Unequal crossing over - Transposons

Exogenous factors

cause of mutation (induced mutations) - Environmental chemicals - Radiation

loss of function mutation

causes a complete or partial loss of function

lethal mutation

causes premature death

gain of function mutation

causes the appearance of a new trait or function or causes the appearance of a trait in inappropriate tissue or at an inappropriate time

nonsense mutation

change a sense codon into a nonsense codon, causing premature termination of translation

silent mutation

change a sense codon into a synonymous codon, leaving the aa seq. of the pro unchanged

missense mutation

changes a sense codon into a different sense codon, resulting in the incorporation of a different aa in the pro.

neutral mutation

changes the aa seq. of a pro w/o altering its ability to function

Base substitution

changes the base of a single DNA nucleotide

forward mutation

changes the wild type phenotype to a mutant phenotype

Deamination can occur by

chemical change to DNA bases Deamination of adenine by Nitrous Acid results in hypoxanthine, which base pairs with cytosine: AT→ GC transition

deletion

deletion of one or more nucleotides

in-frame deletion or insertion

deletion or insertion of a multiple of three nucleotides

Nucleotide excision repair (NER)

employed if bulky damage present (e.g. thymine dimers) remove oligonucleotide • Thymine dimers >> distortion of the DNA helix • The NER system: 1. Finds the distortion 2. Separates the DNA strands 3. Stabilizes the separated strands 4. Cleaves the phosphodiester DNA backbone on both sides of the damage 5. Fills in the gap 6. Ligates the backbone together

the ames test experiment

experiment: how can chemicals be quickly screened for their ability to cause cancer? • Start with his- bacteria. These bacteria can't survive in the absence of histidine. • Mix the bacteria with a mixture of liver enzymes, which can convert some compounds into potential mutagens • Mix the experimental group with the compound being tested for mutagenicity. The control does not get mixed. • Plate the bacteria on media lacking histidine • Do bacteria grow? • If more colonies grow on the experimental plate than the control plate, then the chemical caused his- to his+ mutations • If growth resembles that on the control plate, the chemical did not increase the mutation rate.

Mismatch repair

fixes incorrectly matched base pairs • How are mismatches generated? By misincorporation of standard bases (e.g. "wobble") • The most important role of mismatch repair is as a "last chance" errorcorrecting mechanism in replication • Mismatch repair fixes incorrectly matched base pairs: a segment of DNA with base mismatch is excised, followed by repair synthesis

Transposons (Jumping Genes) are mutagenic by

i. disrupting gene function ii. due to recombination between repeated transposons iii. due to imprecise excision

Suppressor mutations

in the same gene (intragenic), or in a different gene (intergenic)

Many repair mechanisms overlap

in their ability to fix the same types of errors (redundancy)

Indels that do not change the reading frame are called

in-frame insertions or deletions.

Wobble leads to

incorporation errors & mutation (Rare events) Non watson and crick base pairing: Thymine-guanine wobble Cytosine-adenine protonated wobble After next round of replication, the DNA of one daughter will have a TA base pair, the other will have a CG base pair

frameshift mutation

insertion or deletion that alters the reading frame of a gene

conditional mutations

no phenotypic changes under permissive conditions, but phenotype apparent under restrictive conditions

Mutations can be [Mutation hot spots]

nonrandom with respect to position in a gene or genome

Mutations are called dynamic mutations because

of the extraordinary genetic instability of the region of DNA involved

Most DNA repair mechanisms use

one DNA strand as the template ( importance of incorporated vs replicated error)

Real back mutations ("reversions") are

rare

Deletions

removal of 1 or more basepairs of DNA

deamination mutagen

removal of an amino group on a base, induced by nitrous acid

AP endonuclease system

repairs nucleotide sites at which the base has been lost or removed • Purines in DNA prone to hydrolysis, which leave a site that is lacking a purine base = an apurinic site • Both apyrimidinic and apurinic sites (AP sites) repaired by system that depends on an enzyme called AP endonuclease

expanding nucleotide repeats

repeated sequence of a set of nucleotides in which the number of copies of the sequence increases

Mutations can involve large sections of chromosomes or

single base pairs.

Two pathways for mutagenesis by 5-bromouracil

slide 29 for image A. 1) incorporations of Bu in its mutagenic pairing configuration is rare 2) normal pairing of an already incorporated Bu is frequent 3) the result is a GC to AT transition B. 1) incorporation of Bu on its nonmutagenic pairing configuration is common 2) mispairing of an already incorporated Bu is rare 3) the result is a AT to GC transition

suppressor muation

suppresses the effect of an earlier mutation at a different site

intragenic suppressor mutation

suppresses the effect of an earlier mutation w/n the same gene

intergenic suppressor mutation

suppresses the effect of an earlier mutations w/n a different gene

How to Reverse Mutations?

suppressor mutations, intragenic suppressor, intergenic suppressor

Nonsense suppressors

tRNAs with mutated anticodons

Reverse mutations change

the mutant allele back to the wild-type allele

Suppressor mutations do not change

the original mutation - they affect a different gene or a different nucleotide in the same gene wild type: A+B+ Red eyes--> forward mutation of A- = Mutation A-B+ white eyes Mutation A-B+ white eyes ----> reverse mutation of A- = wild type A+B+ red eyes Mutation A-B+ white eyes ----> suppressor mutation B- = mutations A-B- Red eyes

Inverted repeats are recognized by

transposase

what is the molecular basis of genetic instability ?

trinucleotide repeat expansion

Excision repair

works on a wide variety of damaged DNA • Excision repair: stretch of damaged DNA strand removed from duplex molecule • replaced by resynthesis using the undamaged strand as a template

Mutation

• A heritable change in DNA sequence, such as: - Point mutation ("gene mutation") - Rearrangement ("chromosome mutation") - Duplication ("chromosome mutation") - Deletion or deficiency (gene or chromosome mutation) • Inherent property of DNA • Either spontaneous or caused by mutagens

Trinucleotide repeats

• Frequently occurring, spontaneous mutations in certain families • Mutations are called dynamic mutations because of the extraordinary genetic instability of the region of DNA involved • Normal number of repeats is usually small and variable • The molecular basis of genetic instability is a trinucleotide repeat expansion • Occurs due to replication slippage

temperature-sensitive mutations

• Mutants show phenotype only at certain temperature • Temperature changes protein conformation or stability • Can sometimes inactivate and reactivate rapidly = on/off switch for protein function

Transposons (Jumping Genes) are mutagenic: (iii) due to imprecise excision

• Once a transposon has integrated into a DNA site, it can jump back out (excise). • Transposon excision usually leaves a "scar" of the two flanking direct repeats - Precise excision: transposon jumps and one flanking direct repeat is deleted, "healing" the gene (very rare) - Imprecise excision: transposon jumps and repair deletes part of the adjacent chromosomal DNA

eyeless/pax6 encode highly conserved proteins that can function in very different animals

• Vertebrate pax6 is orthologous to eyeless in flies • Mouse pax6 mutants do not form eyes • Expressing the mouse pax6 gene in eyeless mutant flies rescues eye development!!!