Lecture 5/6: Mutation Causes and DNA Repair Learning Objectives

Define spontaneous and induced mutations and explain the difference between them

1. Mutations can occur spontaneously:

2. Errors in DNA replication (strand slippage, base mismatches)

3. Reactivity of the cellular environment (depurination, deanimation, oxidation)

• Mutations can also be induced:

1. Associated with some external factor that changes DNA

2. Particularly useful in model organisms

List the types of spontaneous mutations and explain the mechanism that causes them

• Formation of abasic sites:

1. Hydrolysis reaction will remove the nitrogenous base from the rest of the nucleotide

2. Most commonly called depurination

3. Occurs multiple times an hour in cells

4. The abasic site cannot specify a complementary base

5. Can result in removal of a nucleotide pair during replication

6. Can result in replacement of abasic nucleotide with incorrect nucleotide

• Deamination of nucleotides:

1. Removal of an amino group from the nitrogenous base of the nucleotide

2. Note: thymine is the only base that cannot be deaminated

3. Commonly results in the substitution of one nucleotide for another

4. Remember, only one cell will get the mutation, the other cell will have DNA with the wildtype sequence

• DNA replication errors:

1. Occasionally, the wrong base is added during DNA replication

2. Nitrogenous bases have different forms called tautomers

3. Similar chemical forms that interconvert continually

4. These do not follow standard Watson-Crick base pairing rules

5. May result in substitution mutations in one strand of the helix

6. DNA strand slippage can occur during replication. AKA replication slippage. Well characterized mechanism of unstable trinucleotide repeats

7. DNA polymerase has a tendency to stall in repetitive regions of DNA. A loop will form either on newly synthesized strand or template strand

List the types of mutations that induce mutations and explain the mechanism of each

• Induced mutations are caused by an external environmental factor

• A mutagen is any physical or chemical agent that raises the frequency of mutations above the spontaneous rate

• Examples of mutagens include multiple types of chemicals and radiation. Ex. Radium is a naturally occurring radioactive element. Decaying radium can excite fluorescent materials and make them glow. When ingested, radium mimics calcium and accumulates in the bones

• Base analogs:

1. Molecules with a similar chemical structure to nitrogenous bases

2. Can be incorporated into DNA during replication. Ex: 5-bromouracil can pair with A or G

3. Frequently results in substitution mutations

• Alkylating agents:

1. Modify the nitrogenous base through the addition of alkyl groups, methyl groups, or ethyl groups Ex: Ethylmethylsulfonate, mustard gases

2. Most commonly results in substitution mutations

• Intercalating agents:

1. chemicals that can slide between base pairs and distort the double helix. Ex: Proflavine, acridine orange

2. Will cause nucleotide insertions and deletions

3. Distortion of the helix disrupts action of DNA polymerase

• Adduct forming agents:

1. Chemicals that add an additional chemical moiety to DNA via a covalent bond

2. Includes multiple environmental hazards, including chemicals in cigarettes

3. Can inhibit DNA replication and transcription or cause other errors

• Two types of radiation can change DNA:

1. Ionizing radiation-removes an electron, X-rays and gamma rays

2. Ultraviolet radiation

3. Both either directly damage DNA or excite neighboring molecules that will cause the damage

• Ionizing radiation:

1. Generates reactive oxygen species (ROS)

2. Superoxide radicals, hydrogen peroxide, hydroxyl radicals

3. Ionizing radiation can enhance formation of abasic sites

4. Ionizing radiation can induce breaks in phosphodiester bonds

Ultraviolet (UV) radiation:

1. Causes the formation of pyrimidine dimers

2. Also called thymine dimers

3. Two adjacent pyrimidines within the same DNA strand become covalently linked

4. Distorts the shape of the DNA helix

Connect the causes of mutations to the functional effects on the gene products

Use previous information

Explain why the rate of mutation is different between spontaneous and induced mutations. Explain why induced mutagenesis is a useful experimental tool

• The rate of spontaneous mutations is quite low. Rough calculation: 1 new mutation affecting phenotype could arise in every 3-20 human gametes

• BUT, the rate of mutation can vary among genes. Ex: Larger genes have more nucleotides to hit

• In general, rates of forward mutation are higher than rates of reverse mutation. Easier to break a gene than to fix it

• Among humans, higher mutation rates are found in sperm as compared to eggs. Genome sequencing shows children have, on average, 60 base pairs that are different from parents. Most are inherited from paternal donor, as more cell divisions are needed to make mature sperm

• Genetic analysis frequently utilizes mutagens to create mutations at higher rates than would naturally occur. Nature is too big and too slow to wait for spontaneous mutations to appear in a phenotype. Induced mutagenesis can result in mutations in multiple genes within one organism

List the types of DNA repair systems and explain the mechanism of function

• Proofreading and editing by DNA polymerase:

1. In addition to adding nucleotides, the polymerase can detect when the incorrect nucleotide is added and remove it

2. Called the 3’-5’ exonuclease activity

3. DNA polymerase can re-copy the portion with the excised nucleotide

4. Improves fidelity of copying 100-fold

5. DNA proofreading is not perfect and cannot repair nucleotides that have some type of damage. Multiple other systems are in place in cells to prevent the sequence change from becoming a heritable mutation. In general, the systems are based on the type of damage they repair

• Base Excision Repair (BER):

1. Removal of an incorrect or damaged nucleotide

2. Damage that does not significantly distort the shape of the double helix

3. Corrects oxidative, deamination, alkylation, and abasic damage

4. Critically important to remove uracil from DNA

5. Repair is mediated through DNA glycosylases

• Nucleotide Excision Repair (NER):

1. Removal of multiple nucleotides from the DNA

2. Damage that does significantly distort the shape of the double helix

3. Corrects pyrimidine dimers and DNA adducts

4. Repair is mediated through protein complexes

5. Eukaryotes include the XP proteins, which cause xeroderma pigmentosum (XP) when genes are mutated

• Double Strand Break Repair (HR):

1. Homologous recombination repairs DSBs without gain or loss of nucleotides

2. Cells undergoing meiotic recombination use the homologous chromosome as a template

3. In mitotic cells, DSBs are acquired rather than initiated

4. But, many proteins are the same to repair

5. Template DNA can be the sister chromatid or the homologous chromosome (but harder to find this in mitosis)

• Double Strand Break Repair (NHEJ):

1. Non-homologous end-joining (NHEJ) repairs DSBs but does not rely on homology

2. May result in insertions or deletions of nucleotides

3. If more than one DSB is present in the genome, wrong ends may be joined together

4. Multiple proteins are needed to recognize the break, protect the ends from degradation, then join them back together

• Mismatch Repair (MMR):

1. Corrects base mismatches and strand slippage loops that occur during replication but are not immediately corrected

2. Requires identification of the new strand and removal of multiple nucleotides surrounding mismatch

3. Unclear how the new strand is identified in eukaryotes

4. Bacteria rely on methylation states to distinguish the template strand from the newly synthesized one. Called methyl-directed MMR

5. Prokaryotic and eukaryotic MMR use a complex of proteins. Mut class is required for pathway

6. Mutations in MMR proteins associated with Lynch Syndrome. Increases the risk of developing multiple types of cancer, including colon, endometrial, stomach, pancreatic, etc.

• Error-Prone Repair Systems:

1. Sometimes, the repair systems previously described are not sufficient to repair damage. Ex. many thymine dimers are present than can be repaired in the time before cell division would occur

2. Dangerous to the cell because division can be inhibited and cell will die

3. Translesion synthesis (TLS) uses “sloppy polymerases” to replicate through damage so cell can divide. Uses TLS polymerases

4. TLS polymerases have multiple characteristics that permit replication through damage. Larger active site to accommodate damage. Lack of 3’-5’ proofreading activity. Can only add a few nucleotides before dissociating from template. Use of TLS is a method of last resort

Explain why mutations in genes that encode components of DNA repair systems can increase rate of mutations in those cells

Mutations in genes encoding DNA repair components accelerate the cell's overall mutation rate because they disable the cellular "spell-checkers" and restoration machinery that normally fix routine DNA damage

Apply concepts of DNA mutation and repair to examples not presented in class

• Cockayne Syndrome (ERCC6 / ERCC8 Mutations):

  • DNA Damage Cause: Endogenous oxidative lesions or UV radiation stall RNA Polymerase II during active gene transcription.

  • Repair Defect: Defective Transcription-Coupled Nucleotide Excision Repair (TC-NER). The cell fails to clear stalled polymerases from actively transcribed genes.

  • Functional Effect: Persistent transcript blockages induce widespread apoptosis in non-dividing cells (like neurons), causing progressive neurodegeneration and premature aging rather than classical skin cancer.

• MUTYH-Associated Polyposis (MUTYH Gene Mutations):

  • DNA Damage Cause: Cellular metabolism generates reactive oxygen species, oxidizing guanine into 8-oxoguanine (8-oxoG). During replication, DNA polymerase mispairs adenine ($\text{A}$) opposite 8-oxoG.

  • Repair Defect: Defective Base Excision Repair (BER). MUTYH normally codes for an adenine DNA glycosylase that excises the mispaired $\text{A}$ opposite 8-oxoG.

  • Functional Effect: If the adenine is not excised before a second round of replication, $\text{A}$ pairs with $\text{T}$, permanently converting the original G-C pair into a T-A transversion mutation (often mutating driver genes like APC, leading to colorectal polyps).

• Ataxia-Telangiectasia (ATM Gene Mutations):

  • DNA Damage Cause: Ionizing radiation or metabolic stress creates double-strand breaks (DSBs).

  • Repair Defect: Defective DNA Damage Signaling Checkpoint. ATM codes for a master kinase that detects DSBs and phosphorylates target proteins (like p53 and BRCA1) to halt the cell cycle and recruit repair machinery.

  • Functional Effect: Loss of ATM prevents cell cycle arrest at $G_1/S$ or $G_2/M$ checkpoints. Damaged cells continue dividing with unrepaired double-strand breaks, driving massive chromosomal translocations, cerebellar neurodegeneration, and high lymphoma risk.