Exam 3: MCB

mutations

What accounts for the greatest number of heritable changes in an organism's genome

DNA replication

-it is however still the most reliable

How often do errors occur in DNA replication

• Frequency of mutations in E.coli 4 mutations/100 cell decision

• Average in all organisms is one mistake per billion nucleotides 

• In vitro, e coli makes 400 mistake/cell division 

Why are mistakes in transcription or translation not as critical as in DNA replication

1. Many copies of RNA and then protein produced 

2. RNA and proteins are not heritable over multiple generation

Gene naming

Italized, plus or minus superscript (wild type vs mutant), and lowercase 

• gene^- (mutant type)

• gene^+ (wild type) 

Mutagens: What are they? What are they classified as?

Mutagens: agents that cause mutations

Classified: chemical and physical mutagens

Chemical vs physical mutagens

Chemical mutagens: chemical substances may cause changes in the structure of DNA (act by covalently modifying the structure of nucleotides (base does not pair properly))

Physical mutagens: physical agents such as UV light and X ray can damage DNA (alter structure of DNA)

What are the three ways to fix mistakes during DNA replication?

1. Proofreading

2. Methyl Directed Mismatch Repair (MMR)

3. Nucleotide excision repair

Wha is the window time for Methyl directed repair to occur?

Mismatch repair enzymes fix problems in unmethylated new strands. Once DNA is methylated no more repairs can be done because they are unable to identify daughter vs parental strand, and therefore they do not know which base is wrong.

What is the universal process of Methyl directed repair? (7)

Process:

1. MutH: looks for hemimethylated sequences and latches onto one in order to identify which strand is which 

2. MutS: scans the DNA and looks for problems. Identifies the problem. (not its job to know which base is wrong) 

3. MutL: links MutH and MutS together so they can use the knowledge of the problem and which strand is which to solve the problem

4. MutH: uses its endonuclease activity to nick non methylated strand and recruits an exonuclease 

5. MutS: tells exonuclease to stop once it goes right past the problem (exonucleases do not know when to stop on their own).  MutS then recruits DNA polymerase 3

6. DNA polymerase 3: fills in the gap

7. Ligase: seals the nick

Nucleotide excision repair: What is it? What does it fix (4)? What 4 key proteins is it composed of? What 2 other things does it also require?

Nucleotide excision repair: A region encompassing several nucleotides in the damaged strand is removed from the DNA and the intact undamaged strand is used as the template for the resynthesis of a normal complementary strand 

Fixes:

1. UV induced damage

2.  (thymine dimers)

3. Chemically modified bases

4. Missing bases 

Composed of 4 key proteins: UvrA, UvrB, UvrC, uvrB (involved in ultra violet light repair of thymine dimers), Requires: DNA polymerase and ligase  

Process of nucleotide excision repair (5)

1. Two UvrA proteins and one UvrB protein form a complex that tracks along the DNA. The complex can sense the the distorted DNA

2. When the complex identifies the damage site, the two UvrA proteins are released and UvrC bind to UvrB at the cite

3. UvrC protein makes cuts in one DNA strand on both sides of the damaged site

4. After the cuts have been made UvrC is released. UvrD binds to UvrB. UvrD begins to separate the DNA strand and UvrB is released.  The action of UvrD unravels the DNa which removes a short DNA strand that contains the damaged region. UvrD is released

5. After the damaged DNa strand is removed, a gap is left. DNA polymerase fills the gap using the undamaged strand as a template. DNa ligase makes the final covalent connection between the newly made DNA and the  original DNA strand 

Point mutations: What are they? What are the two types of point mutations?

Point mutations: Affects only a single base pair within the DNA causing consequences when genes are expressed 

Two Types:

1. Base substitution

2. Frame shift

Base substitutions: What are they? What can they be classified as (2)? What are the categoriess of base substitutions (3)?

Base substitutions: one base in the template strand is replaced by another base and therefore the complementary base changes too (replace on base pair with another)

Every base pair substitution is either a: 

Transition base substitution: the base did not change families (purines or pyrimidine)

Transversion base substitution: the base did change families 

Categories of base substitutions when they affect protein coding region:

• Missense

• Nonsense

• Same sense

Frame shift: What is it? What is its effect on the length of the protein product?

Addition or removal of a small number of bases not divisible by three. Disrupts the normal reading frame in the mRNA and produces a different amino acid sequence downstream from mutation. 

• Effect on protein product: usually results in premature appearance of stop codon 

Chromosomal level mutations what does it cause (5)

1. Insertion: Section of DNA (thousands of bases) inserted into DNA

2. Deletion: Section of DNA (thousands of bases) deleted from DNA

3. Inversion: occurs when a segment of a chromosome breaks off, flips 180 degrees, and reattaches in the reverse order without a net gain or loss of genetic material

4. Duplication: duplicated a region (thousands of bases) of DNA 

5. Translocation: occurs when a piece of a chromosome breaks off and attaches to a different, non-homologous chromosome (typically the end of chromosome)

How do mutations affect Germ line cells?

(egg and sperm cells)

If a mutant human gamete participates in fertilization, all cells of the resulting offspring will contain the mutation.  Additionally the mutation may be transmitted to future generations of offspring (50% chance)

How do mutations affect Somatic cells?

(non sex cells) 

A mutation occurs on a cell that was a precursor for many cells of the adult and therefore, as an adult a patch of tissue contains cells that carry the mutation 

*Mutation Passed to daughter cells in the areas* 

Mosaic: somatic regions that are genetically different from each other 

Categories of base substitutions: Missense, nonsense, same sense

Missense: changes one amino acid codon to another amino acid codon in the polypeptide 

• If amino acid that has been altered is not crucial for the folding or function of that protein or if the amino acid that replaces it assumes those duties in roughly the same way (negative for negative) then protein is likely to retain at least some function or may even be fully functional

• If altered amino acid is critical and the duties are not taken up by amino acid that replaces it, then the protein is altered

Nonsense: changes a normal codon to a stop codon and shortens the polypeptide because termination occurred earlier than expected 

• Result: Null phenotype: protein is truncated and virtually always inactive

Same Sense: the effect of one base ends up still being the same amino acid even though different code (no effect on protein product or function)

Effect of in frame removal of 3 bases

the loss of one amino acid and the rest of protein is unaffected (reading frame not affected) 

Ames test

A way to test and evaluate the ability of a substance to cause mutation 

Test that uses a strain of bacteria that cannot synthesize the amino acid histidine 

• Strain contains a point mutation within a gene the codes an enzyme required for histidine biosynthesis 

• The mutation renders the enzyme inactive 

• The bacteria cannot grow unless histidine has been added to growth medium

• A second mutation may correct the first mutation thereby restoring ability to synthesize the histidine

The ames test monitors the rate at which this second mutation occurs and thereby indicates whether an agent increases the mutation rate above the spontaneous rate 

Cystic Fibrosis

characterized by a faulty protein (CFTR) because of the Deletion of codon 508. 

• Result: Mutant protein does not fold properly and therefore never gets to cell membrane which causes Cl- to accumulate in cells which makes an osmotic imbalance and then the mucus layer dehydrates interfering with breathing & digestion

Sickle cell amnemia

Missense: codon 6 changed amino acids

• Result: a single base pair substitution changes charge of protein casing sickle cell amnesia 

Phenylketonuria (PKU)

Missense: Phenylalanine changed to tyrosine 

• Result: newborns screened to prevent severe mental impairment; can be prevented by special diet. Causes a buildup in phenylalanine and low amount of tyrosine (normal growth and skin pigment inhibited)

Lymphomas and Leukemias

caused by translocations

Oncogenes

activated and become cancers through duplications 

Fragile X syndrome and Huntington's disease

caused by duplications of short repeated sequences

Haemophilia A

loss of clotting factors caused by insertions

Duchenne's muscular dystrophy

caused by a deletion 

Silent missense possible effect

if inversions don't involve the loss DNA they are often silent, but have been known to cause infertility

Cytoskeleton: What is it? What is the use? What is the location?

Definition: Dynamic system of proteins fibers of various sizes (can reorganize the cytoskeleton as needs of cell change)

Use: providing physical support and allows for movement (ex: phagocytosis, mitosis)

Location: found in the cytosol and in the nucleus along the inner nuclear membrane 

Actin filaments (micro filaments): What is the diameter? What are the building block? What is the directionality? Binding site characteristic? What are 4 other characteristics?

Diameter: 7nm

Highly folded

Building blocks: Globular proteins: G actin

Directionality: plus (barbed) and minus (pointed) end 

Binding site: Has a nucleotide binding site that can accommodate ATP or ADP 

Characteristics:

• Highly conserved 

• Very abundant 

• Thin and long  

• Highly folded

Polymerization process of microfilaments (actin filaments)

Actin monomers polymerize “head to tail”: made one monomer at a time 

1. nucleation (forming a small group) collision of G actin to stick together. Initiation of an actin filament by association of three or four actin monomers

• Nucleation step is limiting rate step 

2. This forms a nucleus of actin (not an organelle) 

• F actin filament (polymer version) 

3. Polymerization and depolymerization occurs at both ends and is reversible

• polymerization is favored at plus ends and depolymerization is favored at the minus end because that way it is physically easier (dependent on concentration)

Actin binding proteins (ABP): What are they and what do they allow? What are the 6 functions it carries out?

Actin-binding proteins (ABPs) regulate how actin filaments are assembled, maintained, and organized. They allow microfilaments to form different structures for different cellular functions.

• Building-block availability proteins: Control the availability of actin subunits, influencing filament growth and length.

• Actin-severing proteins: Cut actin filaments into smaller pieces.

• Bundling proteins: Hold parallel actin filaments together in tight bundles.

• Cross-linking proteins: Connect actin filaments into flexible networks that provide support while allowing movement.

• Capping proteins: Attach to filament ends to prevent or regulate the addition and removal of actin subunits.

• Anchoring proteins: Attach actin filaments to specific structures within the cell.

What two structures do ABP (actin binding proteins) allow for microfilaments to be assembled in?

Actin bundles

• Make microvilli, temporary finger like projections of plasma membrane, that increase surface area 

• Held together by actin filaments and bundling proteins 

• Actin capping proteins at tips to prevent polymerization or depolymerization 

• Anchored in the terminal web, a network of actin filaments beneath the cell's surface that helps support the microvilli.

Networks 

• Provide the structural stability but are more flexible than bundles 

• Terminal web of actin 

• Formed when actin filaments are connected by flexible cross-linking proteins 

• Cell crawling 

Four functions that actin and myosin work together to do

• Cell division

• Cell crawling

• Movement of some vesicles

• Muscle contraction

Myosin: what is it? What is its relation to actin filaments

Def: Myosin is a molecular motor that converts chemical energy from ATP into mechanical energy to generate force and movement.

Relation: Motor Protein for Actin Filaments

What is myosin’s II goal?

goal is to move toward the plus end of an actin filament and interact with actin to generate force

What is filament sliding?

Filament sliding occurs when myosin pulls actin filaments, causing them to slide relative to one another. This produces movement and force without the filaments themselves needing to shorten.

Process of filament sliding

1. ATP binds to myosin, causing it to detach from actin.

2. ATP is hydrolyzed and ADP and phosphate are now in the binding site, energizing myosin and changing its shape.

3. phosphate is ejected

4. head group is repositioned and is closer to the plus end 

5. Myosin binds to actin again.

6. This triggers the Power stroke: Myosin changes bends and pulls the actin filament 

7. ADP released and there is nothing in the binding site and the cycle repeats as long as ATP is available.

Spontaneous vs induced mutations

Spontaneous:

result from abnormalities in biological processes.  Spontaneous mutations reflect the observation that biology is not perfect 

Induced: 

Caused by environmental agents that enter the cell and alter the structure of DNA like chemical and physical mutagens.  They cause the mutation rate to be higher than the spontaneous mutation rate

Xeroderma Pigmentosum (XP) and DNA Repair

• Cause: An inherited mutation in a gene responsible for DNA repair.

• DNA damage: UV radiation from sunlight damages DNA by creating thymine dimers.

• Repair defect: Cells cannot properly repair this UV-induced DNA damage.

• Result: Damage accumulates, causing mutations that greatly increase the risk of skin cancer and extreme sensitivity to sunlight.

Key takeaway: Defective DNA repair allows UV-induced mutations to accumulate, leading to xeroderma pigmentosum (XP).

Lederberg Replica Plating Experiment

Purpose: Determine whether bacterial mutations happen before or after exposure to an antibiotic.

Steps:

1. Bacteria were grown on a plate to form colonies.

2. Colonies were transferred using velvet onto new plates, including one containing an antibiotic.

3. The same colonies survived on the antibiotic plate in the same positions, showing that some bacteria were already resistant.

Conclusion:

• Antibiotic resistance mutations occur randomly before exposure to the antibiotic.

• The antibiotic selects for resistant bacteria rather than causing the mutations.

• Resistant bacteria survive and reproduce, while nonresistant bacteria die