SB 10: Ch 7.1-7.3 - Mutation & DNA Repair

What is a forward mutation?

• A mutation that changes a wild-type allele of a gene to a different allele, resulting in a novel mutant allele

• Most common

Reverse mutation?

• A mutant allele reverts back to wild-type

• Reversion

Substitution? What are the two types?

• Occurs when a base at a certain position in one strand of the DNA molecule is replaced by one of the other three bases

• After DNA replication, a new base pair will appear in the daughter double helix

• Transition: Purine for purine, pyrimidine for pyrimidine

• Transversion: Purine for pyrimidine, pyrimidine for purine

Three types of point mutations?

• Substitution

• Deletion

• Insertion

Spontaneous Mutation Rate

• A new mutation could arise in every 3-17 gametes

• Some genes mutate more often than others

• Spontaneous mutation rates are low and vary among different genes and organisms

• The more cell divides, the more likely it is that mutations will accumulate in their genomes

How do spontaneous mutations arise randomly?

• Fluctuation Experiment

  • Fluctuations in the numbers of resistant colonies growing in different petri plates showed that resistance is not caused by exposure to bactericides

• Replica Plating

• Results of both show that resistance mutations arise randomly in bacteria cells prior to bactericide exposure

Genetic Screen vs. Genetic Selection

• Screen: everything grow and you look for mutants

• Selection: only letting mutants grow

Mutations that come from natural processes: Depurination

DNA alteration in which a purine base, either A or G, is hydrolyzed from the deoxyribose-phosphate backbone

• Results in apurinic site that cannot specify a complement base

• DNA replication process inputs a random base > mutation in the new complementary strand

Mutations that come from natural processes: X-Rays

• X-rays break the sugar phosphate backbone and split DNA into smaller pieces

• Creates double-stranded breaks

• May be ligated back together incorrectly

• Ionizing radiation

Mutations that come from natural processes: Ultraviolet Light

• UV radiation causes adjacent Ts (thymines) to form dimers, which lead to substitutions when DNA is replicated

• Kink in strand

• Disrupts replication

• Nonionizing radiation

Deamination & Oxidation

• Deamination: removal of an amino group from C causes a transition mutation after DNA replication

• Oxidation: irradiation causes the formation of free radicals (oxygen molecules with unpaired election) that can alter individual bases

  • Altered guanine (G) base into GO paired with A creates transversion

Mistakes in DNA replications, how do they occur and how rare?

• An incorrect base during replication (ex: a C opposite an A instead of a T) will lead to a mutant base pairing in the next replication cycle

  • One daughter strand with A:T, one with mutant G:C

• Extremely rare

What is the proofreading function of DNA Polymerase?

• Error rate of 1 in ever 10^6 bases copied

• Polymerase molecules have a proofreading/editing function in the form of a nuclease which becomes active during a mistake

• 3’ to 5’ exonuclease recognizes a mispaired base and excises it allowing polymerase to copy the NT correctly on the next try

• Improves fidelity

How does tautomerization lead to mistakes by DNA polymerase?

• Tautomerization: A chemical compound can rapidly convert into a structural isomer via movement of an H, interconvert continuously

• Each base has two tautomers (similar chemical forms)

• Usually the equilibrium between the tautomers upholds the A:T and G:C pairing

• However, if a base in the template strand is in its rare tautomeric form when DNA polymerase arrives, the wrong base will be incorporated in the new strand as the rare tautomers pair differently than their normal forms.

Unstable triNT repeats

• Certain trinucleotide repeats, ex: CGG, CAG, CTG, are repeated consecutively, making them unstable

• Expansion of the repeats beyond a certain number have been linked to disease-causing alleles

• Why: DNA polymerase can lose its place when replicating repeats (slipped mispairing)

• More repeats = higher probability of expansion or contraction

  • Depends on whether the newly synthesized strand or template strand “slips”

Trinucleotide repeat Diseases

• PolyQ disease

  • Q = amino acid glutamine

  • repeated triplet = CAG

  • Disease allele w too many triplet repeats = abnormal protein (disease)

  • Gain of function, dominant negative, Huntington’s

  • Mutation affects nature of gene product

• Non polyQ disease

  • CGG, CTG, GCC, GAA

  • No protein/decreased levels of protein

  • Loss of function, X-linked recessive, Fragile X Syndrome

  • Mutation affects amount of gene product

Larger repeat numbers means more instability, what does this lead to some alleles being?

• Pre-mutation alleles

• Alleles with intermediate numbers of trinucleotide repeats that are now highly likely to expand or contract during replication (and become mutant)

• Ex: heterozygote females/hemizygous males for Fragile X, normal females heterozygous for pre-mutation alleles are likely to produce gametes with disease-causing alleles

Mutagens

• Any physical or chemical agent that raises the frequency of mutations above the spontaneous rate

  • Radiation, chemicals, infectious agents

• Use mutagens to produce mutations for studies

• Example: X rays break backbone of DNA, mutations can occur during DNA fragmentation and improper repair/ligating (small deletions)

• Mutations can occur during replication, recombination, or repair

Chemical mutagens

• Base analogs: similar, can replace an actual base and have tautomers, which can lead to substitutions

• Intercalators: flat molecules that sandwich themselves between successive base pairs and disrupt replication > deletions/insertions of base pairs

Mutagens and carcinogens?

• Usually mutations that occur in the germ line are only significant, because they can be passed down

• However, mutations in somatic cells in genes that help regulate cell cycle may lead to cancer (most mutagens are carcinogens)

Ames test for potential carcinogens, how does it work?

• check if a chemical can damage DNA (a mutagen) and potentially cause cancer (carcinogen)

• Scientists use bacteria strain (His-) that has a genetic defect, cannot make food/amino acid histidine on its own

• Place bacteria on petri dish without histidine, normally wouldn’t survive

• Add tested chemical + liver enzyme extract from rat (sometimes mammals livers change harmless chemicals into active mutagens)

• Count colonies:

  • Safe/negative: few/no colonies because defect isn’t fixed

  • Mutagen/positive: explosion of colonies because chemical caused reverse mutation which fixed defect, allowing bacteria to make histidine

  • Reverse mutation proves that chemical is a mutagen (has the power to mutate and damage DNA)

DNA base damage can be reversed - alkyltransferase

• Enzyme systems exist to reverse NT alterations

• Methyl/ethyl groups mistakenly added to guanine, this enzyme can remove them to recreate the original base

DNA base damage can be reversed - photlyase

• Recognizes the thymine dimers produced by UV ray exposure and reverses damage by splitting the chemical linkage between them

• Only works in the presence of light, associates with a small molecules (chromophore) that absorbs light in the visible range, enzyme uses energy captures by chromophore to split dimers

• Mechanism is called light repair or photo repair

Homology-dependent repair (damaged bases can be removed + replaced)

• Remove a small region from the DNA strand containing the altered NT, use the other strand as a template to resynthesize the region removed

• Shows the advantage of double-stranded complementary DNA structure

Type 1: Base excision repair

• Type of homology-dependent repair mechanism

• enzyme DNA glycosylases cleaves an altered nitrogen base from the sugar of the nucleotide, releases base and creates apurinic/apyrimidinic (AP) site (different glycosylases enzymes cleave specific damaged bases)

• enzyme AP endonuclease makes nick

• DNA exonuclease makes wider gap

• DNA polymerase fills gap

• DNA ligase seals

• No RNA primer needed because nick/gap leave behind exposed 3’ OH group

• Uracil example on slides

Type 2: NT excision repair

• Type of homology-dependent repair mechanism

• Used when cell lacks a DNA glycosylase to recognize problematic base

• Depends on enzyme complexes containing more than one protein molecule (UvrA, UvrB, UvrC)

• A+B complex patrols DNA for irregularities, detecting lesions like thymine dimers; B+C cuts damaged strand in two places, leaves gap in region of damaged strand

• Filled by DNA polymerase, sealed by DNA ligase

What can double-stranded breaks lead to?

• Usually by X-rays

• Chromosomal breakage can lead to point mutations and large deletions/other arrangements

Repair double-stranded breaks 1: Homologous recombination (HR)

• Uses complementary base pairing to repair breaks accurately with no loss or gain of NTs

• Exonuclease chews back damaged DNA strand, strand invasion allows repair

• Same mechanism as meiotic recombination, but now mitotic and usually uses sister chromatid as template (not homo cz) so that no recombination takes place (since the broken chromatid + template chromatid are identical)

• Occurs between sister chromatids during G2 of interphase

• Finding a homolog is inefficient usually since this is mitotic cells

Repair double-stranded breaks 2: Non homologous end joining (NHEJ)

• Brings together DNA ends that were not previously adjacent to each other, a few base pairs can be lost or added improperly in the process

• Important for breaks occurring G1 phase as there is no sister chromatid at this point to do HR with

• Proteins bind to DNA ends of break site, protect them from nuclease degradation, bridging them for DNA ligase

Problems with NHEJ/inaccuracy?

• Does not involve DNA homology, can join together any ends (other than telomeres), if multiple breaks occurred, could potentially join together the wrong ends, causing inversions/large deletions (cz rearrangements)

• DNA exonucleases/polymerases can act at the broken ends, removing or adding base pairs before ligase seals

Methyl-directed mismatch repair, what does it correct? how does it find errors? in what organisms?

• Corrects errors in DNA replication, specifically by DNA polymerase (when proofreading fails)

• Recognizes mismatched base pairs after replication is done by looking for abnormal bulges and hollows

• To find parent strand and recognize what is the correct template, bacteria methylates parent

• MutL, MutS, MutH, DNA exonucleases, DNA polymerase, DNA ligase

• Prokaryotes!

Emergency repair system in bacteria - SOS System

• Uses error-prone/sloppy DNA polymerase

• Produced in presence of DNA damage, attracted to replication forks that have become stalled at unrepaired/damaged NTs

• Adds random NTs and allows that cell to continue dividing into daughter cells, which usually carry new mutations

• Mutagenic effect of mutagens depends on/is enhanced by the SOS system

Microhomology-mediated end joining (MMEJ)

• Double-stranded break emergency repair, last resort

• Similar to NHEJ, exonuclease trims one strand of DNA at each broken end

• Resection exposes single-stranded region of DNA sequence on either side of break to bring ends together

• Large deletions

DNA repair is essential, what happens when there are mutations in DNA repair proteins?

1. Cells of people with xeroderma pigmentosum lack ability to conduct nucleotide excision repair (recessive mutation - homozygotes)

• thymine dimers from UV rays cannot be removed

• severe freckling, eventually skin cancer

2. Breast cancer genes BRCA 1 and BRCA2 specify proteins that function in double-stranded break repair vis HR

• loss of one or both increases risk of cancer