13: Genetic Mutations

Explain the steps involved in the light-dependent repair mechanism... (4)

1) UV irradiation 2) the enzyme photolyase binds to the thymine dimer in DNA (DNA takes an enzyme) 3) cleavage of cross-links --> photolyase is activated by the absorption of blue light 4) release of the enzyme

What are the methods for mutagenesis by UV light? (2)

1) UV irradiation (physical mutagenesis) --> thymidine dimers 2) UV radiation --> pyrimidine dimers

Explain the process of mutagenesis by UV light --> UV irradiation... (5)

1) UV irradiation is a physical mutagenesis 2) one possible route: cytosine --UV + H2O--> cytosine hydrate 3) another possible route --> thymine + thymine --UV--> thymine dimer 4) hydrolysis of cytosine to a hydrate (2) may cause MISPAIRING during replication 5) combining thymine + thymine (3) results in thymidine dimer lesions (thymidine dimers)

Based on intensity, what are the highest energy levels involved in ionizing radiation? (3)

1) X rays 2) gamma rays 3) cosmic rays

What are the chemical mutagens involved in mutagenesis? (5)

1) alkylating agents 2) base analogs 3) acridines 4) deaminating agents 5) hydroxylating agents

What chemical mutagens are mutagenic to replicating DNA and non-replicating DNA? (3)

1) alkylating agents 2) deaminating agent 3) hydroxylating agent

What are the two types of excision repairs?

1) base excision repair 2) nucleotide excision repair

What are the two different categories of chemical mutagens?

1) chemicals that are mutagenic to both REPLICATING AND NON-REPLICATING DNA --> ex: alkylating agents and nitrous acid 2) chemicals that are mutagenic only to REPLICATING DNA --> ex: base analogs and acridine dyes

What are the possible base-pair changes involved in tautomeric shifts? (2)

1) common and stable base forms --> keto and amino 2) keto and amino can be changed into enol and imino form, which are less stable and less common

Explain the process of a direct transversion caused by deaminating agents... (2)

1) cytosine --> thymine 2) cytosine -- NH3, H2O--> thymine

Explain the steps involved in the base excision repair mechanism... (6)

1) deamination of cytosine 2) binding of uracil DNA glycosylase 3) excision of Uracil (U) 4) sugar-phosphate removed by AP endonuclease and phosphodiesterase --> RESULT: missing nucleotide 5) DNA polymerase --> RESULT: single-strand break 6) DNA ligase --> RESULT: site of repair

What was known about mutations during the time of classical genetics vs. now? (2)

1) during classical genetics --> mutations were induced and localized on the chromosomes and identified by the change in chromosome number or structure 2) Now in molecular genetics --> we can determine what the actual mutation is or we can determine the molecular nature of the mutants

Explain the mutation that would result if hydroxylamine is used as a chemical mutagen in mutagenesis... (3)

1) hydroxylamine (NH2OH) does a very specific mutation --> only causes the GC --> AT transitions (a hydroxylating agent) 2) cytosine --Hydroxylamine (NH2OH)--> hydroxylaminocytosine, forms adenine 3) C-G --> T-A transition

Explain the steps involved in the SOS response mechanism in E. coli... (3)

1) if DNA is heavily damaged by mutagenic agents, the SOS response, which involves many DNA recombination, DNA repair, and DNA replication proteins, is activated 2) DNA polymerase V replicates DNA in damaged regions, but sequences in damaged regions cannot be replicated accurately 3) this error-prone system eliminates gaps but increases the frequency of replication errors

Acridine Chemical Mutagens

(aka intercalating agents) wedges, or sandwiches into the bases, and during replication, it interferes with the replication process --> mutagenic to replicating DNA only

What are the most common chemical mutagens found in base analogs? (2)

1) 5-Bromouracil (5-Bu) 2) 2-Aminopurine (2-AP)

Explain the mutation that would result if 5-bromouracil is used as a chemical mutagen in mutagenesis... (4)

1) 5-bromouracil is a base-analog chemical mutagen 2) mispairing with 5-bromouracil promotes A to G mutation 3) 1st step: 5-bromouracil --> adenine base pair (5-bromouracil, keto form --> adenine) 4) 2nd step: 5-bromouracil --> guanine base pair (5-bromouracil, enol form --> guanine)

Explain the process of an indirect transversion caused by deaminating agents... (3)

1) A--> HPX --> C or C--> U --> A 2) adenine --HNO2--> hypoxanthine, combines with cytosine 3) cytosine --HNO2--> uracil, combines with adenine

What are the two different kinds of transversions caused by deamination in mutagenesis?

1) DIRECT --> modified cytosine (5m C) to T 2) INDIRECT --> (A to HPX to C) or (C to U to A)

What are the most common chemical mutagens found in alkylating agents? (3)

1) Di-(2-chloroethyl) Sulfide (mustard gas) 2) ethyl methane sulfonate (EMS) 3) ethyl ethane sulfonate (EES)

Explain the mutation that would result if EMS is used as a chemical mutagen in mutagenesis and combined with guanine... (4)

1) EMS is an alkylating agent chemical mutagen 2) EMS with guanine undergoes alkylation 3) instead of base-pairing with the cytosine, it base-pairs with thymine 4) CG --> TA mutation

What are three examples of disorders that are the results of trinucleotide repeats?

1) Fragile X Syndrome 2) Huntington Disease 3) Spinocerebellar Ataxia

What are the possible results of a nonsense mutation? (2)

1) if the mutation occurs early (if the stop codon occurs early) --> the gene results in the truncation (shorter) of protein and loss of function (TRUNCATED PROTEIN) 2) if the mutation occurs later in the sequence of the gene --> results in the protein loss of function or reduction in function

What are the possible cause(s) for the formation of mutations in regards to the efficiency of the repair mechanisms? (5)

1) light-dependent repair (photoreactivation) --> does not occur in mammals (only in E. coli) 2) excision repair 3) mismatch repair 4) post replication repair 5) error-prone repair system (SOS response)

How does DNA cause mutations?

DNA seldom makes mistakes, but when they do, the change in genetic material is called a mutation

What is this example of? --> Wild Type A+B+ (red eyes) --> Mutation A-B+ (white eyes)

Forward mutation --> wild-type (A+) is changed to mutant phenotype (A-)

What are the most common chemical mutagens found in deaminating agents?

HNO2 (nitrous acid)

What is the molecular basis of mutation?

How do the mutations come about?

DNA Repair Mechanisms

Living organisms contain many enzymes that scan their DNA for damage and initiate repair processes when damage is detected

What are the two types of non-synonymous mutations?

Missense mutations and nonsense mutations

What are the most common chemical mutagens found in hydroxylating agents?

NH2OH (hydroxylamine)

What is this example of? --> wild type (A+B+) red eyes --> mutation (A-B+) white eyes --> Mutation (A-B-) red eyes

Suppressor Mutation --> there is a second mutation in a different location, original phenotype restored

What is the main difference between transition and transversion substitution point mutations?

TRANSITIONS --> often silent (no change in codon or amino acid) TRANSVERSIONS --> often change codon

How does mutagenesis by UV light cause mutations?

UV light causes thymine dimers, which messes up the structure of the DNA helix --> RESULT: replication

Base Excision Repair

a DNA repair mechanism in which pathways remove abnormal or chemically modified bases

Nucleotide Excision Repair

a DNA repair mechanism in which pathways remove larger defects, such as thymine dimers

Light-Dependent Repair

a DNA repair mechanism in which photolyase cleaves thymine dimers --> does not occur in mammals

Error-Prone Repair System (SOS Response)

a DNA repair mechanism that is considered the "hail Mary" of repair systems

Mutation

a change in the genetic material --> the process by which the change occurs

Suppressor Mutation

a second mutation at a different location in the genome --> it compensates for the loss of the original mutation

Back Mutation

a second mutation at the same site --> it can reverse back to the wild-type

Reverse Mutation (Reversion)

a second mutation that restores the original phenotype

Synonymous Mutations (Silent Mutations)

a substitution point mutation in which the genetic code is degenerate --> no change in the codon (amino acid)

Non-Synonymous Mutations

a substitution point mutation in which there is a change in amino acid

Transversion

a substitution point mutation that involves substituting (changing) a purine to a pyrimidine --> also includes substituting (changing) a pyrimidine to a purine

Transition

a substitution point mutation that involves substituting (changing) a pyrimidine to a pyrimidine (cytosines and thionines) --> also includes substituting (changing) a purine to a purine (adenine and guanine)

Nonsense Mutation

a type of non-synonymous substitution point mutation that involves a change in an amino acid into a stop codon

Missense Mutation

a type of non-synonymous substitution point mutation that involves a change of an amino acid into another amino acid --> Ex: sickle cell disease

Substitution

a type of point mutation that involves changing a base pair

How many deaminations per cell division are bases subjected to?

about 10-100 deaminations per cell division

Xeroderma Pigmentosum

an inherited human disease caused by defects in DNA repair --> person becomes very sensitive to UV light, adults have to have full protective gear to go outside because any little bit of light can damage their skin

Frameshift Mutations

an insertion or deletion point mutation that involves single nucleotide insertions or deletions

Mutant

an organism that exhibits a novel phenotype

What are the two different kinds of reversions?

back mutations and suppressor mutations

What chemical mutagens are mutagenic to replicating DNA only? (2)

base analogs and acridines

What is the major effect of insertion and deletion point mutations?

both cause a frameshift (change in the reading frame) --> when you insert the gene, the reading frame changes

Alkylating Agents as Chemical Mutagens

can cause all sorts of mutations --> mutagenic to both replicating and non-replicating DNA

Trinucleotide Repeats

can increase in copy number of the gene and cause inherited diseases --> can cause mutations

In human cells, what are somatic cells linked to?

certain types of cancers

Chromosomal Mutations

changes in chromosome number or structure

What are the two different types of mutations?

chromosomal mutations and point mutations

What is the role of AP endonuclease in the base excision repair mechanism?

cleaves out the area that needs to be removed

How can competition between polymerase and exonuclease cause mutations?

competition between polymerase and exonuclease activities of DNA polymerase can cause occasional MISMATCH errors --> keto vs. enol form interferes with the proofreading

What is this example of? --> (1) wild-type gene: ACA (Thr), AAG (Lys), AGA (Arg), GGT (Gly) --> (2) gene with mutation: ACA (Thr), AGA (Arg), GAG (Glu), GT...(Val)

deletion point mutation --> removal of a base pair (A) caused a frameshift upstream

What is the role of DNA polymerase in the base excision repair mechanism?

edits (adds the correct nucleotide)

What plays a key role in the DNA repair mechanisms?

enzymes

What is the product of normal translation in missense mutations? incomplete translation?

functioning protein --> incomplete protein

What are some examples of physical agents of mutagens?

heat and sunlight

Hydroxylating Agents as Chemical Mutagens

hydroxylamine (NH2OH) hydroxylates the amino group of cytosine and leads to G:C --> A:T transitions --> mutagenic to both replicating and non-replicating DNA

What is this example of? --> (1) wild-type gene: ACA (Thr), AAG (Lys), AGA (Arg), GGT (Gly) --> (2) gene with mutation: ACA (Thr), GAA (Glu), GAG (Glu), AGG (Arg), T

insertion point mutation --> introduction of a base-pair (G) caused a frameshift downstream

Insertion Point Mutations

introduce a base pair or more to the sequence of the gene which case a FRAME-SHIFT downstream

What is the role of nitrous oxide (HNO2) in chemical mutagnesis?

it is a deaminating agent chemical mutagen that causes deamination and transversions

What is the role of glycosylase in the base excision repair mechanism?

it is an enzyme that scans and finds the area that needs to be repaired

How is the nucleotide excision repair mechanism different than the base excision repair mechanism?

it is similar, but it uses an enzyme that makes a cut on both sides of the damage and removes it (uses protein UVrc)

At the time of proofreading, what would be the chemical state if POLYMERASE WINS?

last nucleotide would be base-paired

At the time of proofreading, what would be the chemical state if EXONUCLEASE WINS?

last nucleotide would not be base-paired

Base-Analog Chemical Mutagens

look similar to normal bases, so they can be easily inserted into the DNA chains during replication --> mutagenic to replicating DNA only

What is this example of? --> (1) normal hemoglobin DNA: CTT, mutant hemoglobin DNA: CAT --> (2) mRNA: GAA, mutant mRNA: GUA --> (3) normal hemoglobin --> Glu, sickle-cell hemoglobin: Val

missense mutation --> one bp change (T to A), change an amino acid into another amino acid (Glu to Val)

What kind of substitution point mutations occur in hemoglobin?

missense mutations

What are the possible cause(s) for the formation of mutations in regards to exposure to the mutagenic agents and how they can be induced (the mutagenic agents)?

mutagenesis

Forward Mutation

mutation of a wild-type allele to a mutant allele

Point Mutations

mutations at specific sites in a gene --> a nucleotide change at a specific site

What kind of mutations are responsible for most spontaneous mutations?

mutations induced by transposons

Deaminating Agents as Chemical Mutagens

nitrous acid (HNO2) causes deamination, which can lead to transversions (direct or indirect) --> mutagenic to both replicating and non-replicating DNA

Are all mutations bad? Explain

not all mutations are bad: some can create differences in genomes, producing variability between the genomes --> variability makes evolution possible

UV

not as high energy as X rays or gamma rays (non-ionization radiation)

Germinal Mutations

occur in germ-line cells and will be transmitted through the gametes to the progeny

Somatic Mutations

occur in somatic cells --> the mutant phenotype will occur only in the descendants of that cell and will not be transmitted to the progeny

Spontaneous Mutations

occur without a known cause due to inherent metabolic errors or unknown agents in the environment (transposable elements)

How many transitions and transversions occur for every base pair?

one transition and two transversions

Mutagens

physical and chemical agents that cause changes in DNA, such as ionizing irradiation, UV light, or certain chemicals

Deletion Point Mutations

remove a base pair or more from the sequence of the gene which causes a FRAME-SHIFT upstream --> the frame-shift may result in a premature stop codon downstream or changes in amino acid sequence

Induced Mutations

result from exposure of organisms to MUTAGENS

What causes mismatched base pairs in tautomeric shifts?

results in changes from common (keto or amino) to rare (enol or imino) form

What is the role of DNA ligase in the base excision repair mechanism?

seals the strand

What substitution point mutations occur in the coding region?

synonymous point mutations and non-synonymous point mutations

What is this an example of? --> hydrogen-bonded A:C and G:T base pairs form when cytosine and guanine are in their rare imino and enol forms

tautomeric shift --> A:C and G:T are rare base pairs that have changed from the common and stable base form

What are the reversion mutations based on?

the location of the second mutation

Tautomeric Shifts

the natural chemical fluctuations (the H atoms moving around) --> leads to base-pair changes (mismatched base pairs), and mismatched bases can cause mutations

Mutagenesis

the practice of inducing mutations for experimental purposes --> mutations can be induced with chemicals or radiation

What is life based on?

the replication of DNA

How did Watson and Crick develop the basis for the idea of tautomeric shifts?

they found that the bases in DNA were not static (the H atoms can move from one position to another)

What are the pyrimidines?

thymine and cytosine

What factors can cause thymine dimers? (UV irradiation)

too much sunlight and tanning beds

Most spontaneous mutations are the result of...

transposons

X Rays and Gamma Rays

used for medical purposes, they have high energy and are able to penetrate deep into the tissues (ionization radiation)

Chemical Agents of Mutagens

what cause the DNA to change --> ionizing radiation, UV light, and different chemicals

How are thymine dimers formed?

when combining thymine + thymine with UV light, crosslinks between adjacent thymines form, forming the thymine dimer

Explain the process of inducing a mutation by transposons... (4)

1) most organisms contain genetic elements that are able to "jump" and switch positions 2) when the transposons are inserted into a different gene, they can cause the protein to be non-functional 3) normal activity: transcription then translation produces an active polypeptide gene product 4) if the transposable element jumps inside the gene, the gene segments become separated by the transposable genetic element --> transcription then translation produces an inactive truncated polypeptide with reduced function

Explain the features of base-analog chemical mutagens in mutagenesis... (5)

1) mutagenic to replicating DNA only 2) inserted in DNA 3) base-pair inappropriately 4) because of their similarity to normal bases, they can be easily inserted into the DNA chain during replication 5) they are just dissimilar enough to increase the frequency of the mispairing

What are the methods for mutagenesis? (3)

1) mutations can be induced with chemicals or radiation 2) metagenesis by UV light --> UV irradiation and UV radiation 3) mutagenesis by chemical mutagens

Explain the steps involved in the nucleotide excision repair mechanism... (6)

1) polypeptide trimer containing two copies of UvrA protein and one copy of UvrB protein recognizes and binds to damaged DNA --> RESULT: thymine dimer 2) energy from ATP is used to bend DNA and change the conformation of OvrB protein --> UvrA dimer is released 3) UvrC protein binds to UvrB-DNA and cleaves DNA 5' and 3' to dimer --> RESULT: 5' nick and 3' nick 4) UvrD helicase releases excised oligomer --> UvrC + 12-mer released 5) DNA polymerase replaces UvrB protein and fills in the gap using the complementary strand as a template 6) DNA ligase seals the nick left by polymerase --> RESULT: 12 nucleotides replaced

What are the possible base changes involved in transitions? (2)

1) purine --> purine (A --> G or G --> A) 2) pyrimidine --> pyrimidine (T --> C or C --> T)

What are the possible base changes involved in transversions? (2)

1) purine --> pyrimidine : A-->C, A-->T, G-->C, G-->T 2) pyrimidine --> purine : C-->A, C-->G, T-->A, T-->G

Provide a summary for the products of substitution point mutations that occur in the coding region... (3)

1) silent (synonymous) and no mutation --> no amino acid change 2) nonsense mutation (non-synonymous) --> stop 3) missense mutation (non-synonymous) --> has a change in the amino acid

What are the different categories of mutations? (3)

1) somatic or germinal 2) spontaneous or induced 3) forward or reversed

What are the 3 broad categories of point mutations?

1) substitution 2) insertion 3) deletion

What are the possible causes for the formation of mutations in regards to the accuracy of DNA replication machinery? (4)

1) tautomeric shifts 2) competition between polymerase and exonuclease 3) mutations induced by transposons 4) expansion of trinucleotide repeats

What are the 3 major factors involved in the formation of mutations?

1) the accuracy of DNA replication machinery 2) the efficiency of the repair mechanisms 3) exposure to the mutagenic agents and how they can be induced (the mutagenic agents)

Explain the formation and consequences of pyrimidine dimers... (3)

1) the formation of a thymine dimer by UV light causes the DNA to bulge and lose inter-strand hydrogen bonding 2) during DNA replication the polymerase cannot traverse the dimer and stops, but restarts at another point after the dimer 3) the gap is eventually filled using an error-prone repair system that operates without a template --> incorrect bases are randomly incorporated into the new strand

Explain the process of how the expansion trinucleotide repeats leads to mutations... (4)

1) the human genome has lots of repeat regions (satellite regions) --> simple tandem repeats are repeated sequences of one to six nucleotide pairs 2) there is a specific trinucleotide repeat of three base pairs 3) trinucleotide repeats can increase the copy number of the gene and cause inherited diseases 4) can increase up to 50 times and there is over 50 human genetic disorders linked to this type of mutation

Explain the features of intercalating agent (acridines) chemical mutagens during mutagenesis... (4)

1) they sandwich in between the bases in the DNA 2) mutagenic to replicating DNA only 3) ethidium bromide is an acridine that can be used in the lab when we want to stain our gels --> when we want that chemical to be sandwiched into the DNA so we can see it under a UV light 4) ethidium bromide produces proflavin molecules --> these chemicals mess up the DNA helix and interfere with the replication process

What are the different kinds of substitution point mutations? (4)

1) transition 2) transversion 3) Synonymous mutations (silent mutations) 4) non-synonymous mutations --> Missense mutations and nonsense mutations

What different types of mutations can alkylating agents cause in mutagenesis? (3)

1) transitions and transversions 2) frame-shifts 3) chromosomal structural changes (breaking the chromosome)

What are the most common chemical mutagens found in acridines?

2,8-diamino acridine (proflavin)

How many codons make up the genetic code? What do they code for?

64 codons --> 61 code for amino acids including the start codon (Met, AUG), and the other 3 codons are stop (UGA, UAA, UAG)

What are the purines?

Adenine and Guanine

What is this example of? --> Wild type (A+B+) red eyes --> mutation (A-B+) white eyes --> wild type (A+B+) red eyes

Back Mutation --> there is a second mutation in the same location, it reversed back to the wild-type