Chapter 15: Gene Mutations, DNA Repair, and Transposition Flashcards
Generally speaking, which of the following mutations would most severely affect the protein coded for by a gene?
- a base substitution at the beginning of the gene
- a base substitution at the end of the gene
- a frameshift deletion at the beginning of the gene
- a frameshift deletion at the end of the gene
a frameshift deletion at the beginning of the gene
A frameshift mutation at the beginning of a gene would affect every codon after the point where the mutation occurred. During protein synthesis, incorrect amino acids would be inserted from the point where the frameshift mutation occurred on; the resulting protein would most probably be nonfunctional. For this reason, a frameshift mutation at the beginning of a gene is generally the most severe type of mutation.
What is a mutation?
Mastering Genetics
Any change in the base sequence constitutes a mutation.
A base substitution mutation can occur if the DNA polymerase inserts the wrong nucleotide base as it synthesizes a new strand of DNA. A frameshift mutation can occur if the DNA polymerase leaves out a nucleotide or adds an extra nucleotide to the sequence.
Certain forms of cancer occur because of mutations in DNA sequences that are located in so-called mutational hotspots. These hotspots are locations in the DNA sequence where mutations occur more often than in other places.
Point mutations in DNA sequences can profoundly affect protein synthesis, or they can have no effect at all.
Point mutations can be beneficial to an organism but are more commonly neutral or harmful.
True or False?
Tautomers of nucleotide bases are isomers that differ from each other in the location of one hydrogen atom in the molecule.
True
Nucleotide tautomers differ only in the bonding location of one hydrogen atom.
Which nucleotide will base‑pair with the enol form of 5‑bromouracil?
Guanine
The enol form of 5‑bromouracil forms a base pair with guanine.
Which of the following events could result in a frameshift mutation?
Base deletion
A base deletion would shorten the DNA sequence and change the reading frame of the mRNA.
What fluctuates in the fluctuation test of Luria and Delbruck?
The number of resistant cells from different independent cultures
Although the cultures were treated identically, random mutations gave random fluctuating numbers of resistant bacteria. The numbers fluctuated because the random mutations arose at different times in the different cultures and the resulting cells had varying amounts of time to grow and increase in number.
A point mutation occurs such that a codon is changed from AGA to AGC.
Which term describes this mutation?
Transversion mutation
Transversions involve the substitution of a purine for a pyrimidine or vice versa. In this case, the substitution involved a change from the purine adenine to the pyrimidine cytosine.
Nutritional mutations can be defined as ________.
those mutations that do not allow an organism to grow on minimal medium but do allow growth on complete medium
A class of mutations that results in multiple contiguous amino acid changes in proteins is likely to be which of the following?
frameshift
In general, mutation rates in humans occur in the range of
____ .
per gamete per generation
Why are X rays more potent mutagens than UV radiation?
X rays are of higher energy and shorter wavelength than UV light. They have greater penetrating ability and can create more disruption of DNA.
Change cytosine to uracil and adenine to hypoxanthine by converting an amine group to a keto group
Deaminating Agents
Add a methyl or ethyl group to the amino or keto groups of nucleotides, changing base-pair affinities.
Alkylating Agents
Are incorporated as thymine and adenine, but base pair with guanine and cytosine, respectively.
Include such substances as 2-Amino Purine and 5-Bromouracil
Base Analogs
What is a spontaneous mutation?
Mutations that occur as a result of natural biological and/or chemical processes are considered spontaneous.
In general, mutation rates in humans occur in the range of
10^-6 to 10^-5 mutations per gamete per generation
Mutations that arise in nature, from no particular artificial agent, are called ________.
spontaneous mutations
A mutation that occurs naturally, without exposure to a known mutagen, is called a(n) ________ mutation.
spontaneous mutation
Apurinic sites (AP sites) involve a spontaneous loss of a(n)________ in an intact double-helix DNA molecule.
purine
Which of the following statements about deamination is true?
Deamination is induced by nitrous acid.
The main effect of nitrous acid is to increase the rate of deamination.
What are the consequences of having pyrimidine dimers in DNA?
These dimers distort the DNA structure and result in errors during DNA replication.
True or False?
Thymine dimers can be repaired by Photoreactivation Repair or Nucleotide Excision Repair.
True
Both Photoreactivation Repair and Nucleotide Excision Repair will target UV-induced pyrimidine dimers in DNA.
Which of the following statements regarding Nucleotide Excision Repair (NER) and Base Excision Repair (BER) is true?
Both NER and BER involve the removal of one or more damaged bases by a nuclease.
In both NER and BER a nuclease will target damaged or distorted regions of DNA.
In terms of its involvement in mutagenesis, 5BU is best described as _______.
a base analog that can cause either A-T > G-C or G-C > A-T transitions
In its common form, 5BU can pair with adenine and in its rare form it can pair with guanine.
For 5BU to cause a transition mutation, which of the following must occur?
DNA with incorporated 5BU must replicate.
5BU must undergo a form change, but that is not sufficient to cause transition. The form change must be followed by replication.
True or False?
In its rare form, 5BU pairs with guanine.
True
In its more common form, 5BU will pair with adenine. In its rare form, it pairs with guanine.
Which of the following name two mutagens that would be classified as base analogs?
5-bromouracil and 2-aminopurine
Which enzyme is responsible for proofreading during replication?
DNA polymerase
DNA polymerase performs proofreading functions during replication using its 3’ to 5’ exonuclease capability.
All EXCEPT which of the following are good model organisms for genetic studies?
Humans
Humans do not have any of the characteristics of a good model organism for experimental genetic studies.
All of the following are good model organisms for genetic studies:
E. coli
Mus musculus
Drosophila melanogaster
True or False?
Loss-of-function mutations that eliminate the function of a gene product are also known as null mutations or gene knockouts.
True
True or False?
When treating an organism with a mutagen, although it is possible that homozygous mutations will occur, it is more likely that most new mutations will be heterozygous or hemizygous.
True
True or False?
A missense mutation causes premature chain (protein) termination.
False
Which enzyme is responsible for proofreading during replication?
DNA polymerase
DNA polymerase performs proofreading functions during replication using its 3′ to 5′ exonuclease capability.
True or False?
Bacteria can distinguish between a newly replicated DNA strand and the original template strand because the newly replicated strand is methylated, whereas the original template strand is not.
False
Methylation occurs shortly after replication, so the original template strand is methylated and the newly replicated DNA strand is not.
Which repair system uses the RecA and LexA proteins?
SOS repair
RecA and LexA are active during the SOS response.
Which of the following statements about mismatch repair is false?
It recognizes methylated cytosine.
Mismatch repair recognizes methylated adenine at the sequence GATC, which contains all 4 bases and is a palindrome.
The following statements about mismatch repair are TRUE…
It determines which base in a mismatch is the incorrect one.
It requires the action of a methylase.
It depends on the fact that parental strand DNA is more heavily methylated.
The purpose of the Ames Test is to _______.
test the mutagenic effects of chemicals
The Ames test detects whether a given chemical can cause a reversion mutation in his- bacteria.
In the Ames Test, the appearance of his+ revertants in the presence of a non-mutagenic control compound indicates that _______.
some of the reversion mutations are not caused by the mutagen being tested
His+ revertants on the control plate are the result of spontaneous mutation.
True or False?
Many chemicals are more mutagenic after being processed in the liver.
True
Many potential mutagens are poorly mutagenic until passing through the liver.
True or False?
All compounds that have been found to be mutagenic in the Ames test are also carcinogenic.
False
The Ames test is used as a preliminary screening tool. Not all compounds that give a positive Ames test are carcinogenic.
Why are liver extracts used in the Ames test?
Liver enzymes may activate some innocuous compounds, making them mutagenic.
Some compounds are innocuous until they are activated metabolically by liver enzymes.
Which bacteria grow on the agar plate if the Ames test is positive?
his+ prototrophs
The bacteria used in the Ames test to evaluate mutagenicity are his− auxotrophs. If the Ames test is positive, these bacteria have reverted back to wild type and are his+ prototrophs
Why are spontaneous mutations rare?
They are relatively rare in comparison to induced mutations that are more directed to the physical or chemical properties of DNA.
Assuming one mutational event in a gene, on average, which of the following mutagens or mutational conditions would be expected to cause the most damage to a protein synthesized by such a mutagenized gene: 5-bromouracil, 2-amino purine, ethylmethane sulfonate, or frameshift?
frameshift
In a survey of 240,000 human births, six achondroplastic births were recorded to parents who were unaffected. Given that this form of dwarfism is caused by a fully penetrant, dominant, autosomal gene, what is the mutation rate? (Recall that mutation rate is the number of mutant gametes over the total number of gametes.)
480,000 gamete would be required to produce 240,000 offspring. So, since 6 gametes contain the mutation, the mutation rate would be:
6/480000 = 1.25X10^-5
Mutations =
changes that occur in the DNA molecule.
Fixed mutations can lead to….
altered phenotypes (including diseases).
Mutations
- Point mutations.
- Altered number of copies of repeated sequences.
- Insertion of large segment of foreign DNA into normal gene sequence.
Point mutations:
Definition
Changes to single base (point)
Point mutations are more likely than not to be deleterious
Point mutations:
Causes
Can be spontaneous: arising in absence of known mutagen at some low “rate”
Can be induced by mutagens (agents that increase the rate of mutations)
Transition Mutation
purine to purine
or
pyrimidine to pyrimidine
eg. A to G; C to T
4 possibilities
Transversion Mutation
purine to pyrimidine or pyrmidine to purine
A to T, A to C
G to C, G to T
C to A, C to G
T to A, T to G
8 possibilities
Insertion or Deletion Mutations (aka, Indels, frameshifts)
CONSEQUENCE
Addition or deletion of one or more bases
eg. TAAGCT to TAAGGCT (insertion)
Mutations:
3 main categories
REPLICATION SLIPPAGE LEADING TO INCREASED NUMBER OF REPEAT UNITS
POINT MUTATIONS
INSERTION OF FOREIGN DNA ELEMENT DISRUPTING DNA
Repeat Unit Expansion
Mutations
eg. CGG, CAG, or CTG repeats
Human Diseases:
-Huntington Disease:
repeat expansion in protein coding region (CAG)
-Mytonic Dystrophy:
repeat expansion at 3’ end of transcript (CTG)
-Fragile X Syndrome:
repeat expansion in transcribed region (CGG)
Fragile X Syndrome:
Basics
repeat expansion in transcribed region (CGG)
Fragile X Disease: Tri-Nucleotide Repeat Expansion —> Disease
6-50 copies = Normal
50-200 = Susceptible Lineage
200-1300 = Disease
Fragile X Syndrome
Details
1 in 4000 males, 1 in 8000 female births
Repeat expansion is in 5’ untranslated region (UTR)
Ho: CGG repeats modified, base within & around repeat methylated —–> gene inactivation
Gene is for FMRP (fragile X mental retardation protein)
Normally expressed in brain, binds & potentially transports mRNAs
FMRP Not present
- —> no mRNA transport
- —> no translation of those mRNAs
- —–> improper brain development
Fragile sites and Cancer
Fragile sites now shown to be associated with some cancers
Eg. FHIT gene locus (p arm, chromosome 3) is located within a fragile site on chromosome 3, FRA3B
DNA often found broken and incorrectly fused in cells from tumors
FHIT is a “tumor supressor gene” area of VERY active investigation
~80 or more fragile sites now known
FHIT
tumor supressor gene
FHIT gene locus (p arm, chromosome 3) is located within a fragile site on chromosome 3, FRA3B
DNA often found broken and incorrectly fused in cells from tumors
Mutagen categories
Non-chemical mutagens
Chemical mutagens
Base isomers
Mutagen Consequences
Base replacement
Base alteration leading to mis-pairing
Base alteration leading to non-pairing
X-Rays
Non-Chemical Mutagen
Consequence = Single and double stranded breaks of DNA
UV Irradiation
Photodimers
Non-Chemical Mutagens
Example: Induction of a thymine dimer by UV radiation, leading to distortion of the DNA. The atoms of the pyrimidine ring are shown forming cross-links, becoming a dimer.
Chemical Mutagens
Base analogs
Base alteration chemicals
Intercalating agents
Alkylating agents
eg., EMS
add methyl or ethyl group to base
can lead to G pairing w/T
Alkylating agents
eg., EMS
add an alkyl group (methyl or ethyl) group to base
can lead to G pairing w/T
Intercalating agents
intercalates in DNA stack, can lead to indel —> frameshift
Insert in double helix
- —> Indel
- –>Frameshift mutation
Base analogs
2-aminopurine:
Analog of adenine
Mispairs w/cytosine when protonated.
Leads to A—>G mutation
5-bromouracil:
Analog of thymine
5-BU becomes ionized, pairs
with guanine
alkyl group
An alkyl group is a piece of a molecule with the general formula CnH2n+1, where n is some integer.
For example, a methyl group (CH3) is a fragment of a methane molecule (alkane) (CH4); n = 1 in this case.
The -yl ending means a fragment of an alkane was formed by removing a hydrogen.
Methane, CH4 —->
methyl group, -CH3
Ethane, CH3CH3 —->
ethyl group, -CH2CH3
EMS
Ethylmethane sulfate
Alkylating agent
Tautomers
different chemical forms of DNA bases
Normal=keto
Altered=enol
enol forms can mispair
Tautomeric shift
a migration of a hydrogen atom or proton, resulting in switch of a single bond and adjacent double bond
Base Isomers:
tautomeric shifts
Normal (keto) form of base skewed to rare (enol) form
OR
normal (amino) to rare (imino) of base
Ionization of bases can also lead to shift to rare form
Can lead to mispairing of bases
Tautomeric shifts can lead to mutation
Spontaneous Mutation
LOW RATE
Source of genetic variation
Spontaneous Mutation
Causes
- Oxidative compounds
- Depurination
- Errors in DNA replication
Spontaneous Mutation
Consequences
Base mispairing
Nonpairing or base substitutions
Fixed mutation
Mutation Hot Spots
Some positions/regions more susceptible to mutational changes
Causes:
- replication slippage leading to indels
- 5-methylcytosine sites
5’-Methylcytosine hot spots in E. coli lacI gene
5’-Methylcytosine sites, lose amine group–>
becomes thymine—> pairs w/ adenine
GC—->AT predominantly at these sites.
Mutagens Summary
Background mutation rate
Agents act to increase this rate
- Non-chemical
- Chemical
- Isomerization phenomena
Some sites more susceptible to mutation
AMES Test
Assessment of Mutagenicity
The Ames test screens potential compounds for mutagenicity.
Utilizes 2 Salmonella typhimurium mutant strains, both his- (requires histidine to grow)
Assay measures reversion (mutation) to wild type his+
Compounds modified by liver enzymes prior to test….Why?
Conclusion: Any chemical that significantly increases the number of colonies appearing on the treatment plate is mutagenic, and therefore potentially carcinogenic
Mobile Genetic Elements
DNA moving around within the genome!
Another source of mutations
Transposable Elements
Class I
Insertion Sequences
Can harbor genes
Source of multiple drug resistance transfer
Transposons:
Prokaryotic and Eukaryotic
Transpose through DNA intermediate
“copy and paste” or “cut and paste” movement mechanism
Have inverted terminal repeats
Has transposase enzyme gene
Examples: bacterial IS elements, bacterial transposons, Drosophila P elements, Maize Ac/Ds
Retrotransposons
Class II
“copy and paste” mechanism
Transpose through RNA intermediate, RNA reverse transcribed to dsDNA by reverse transcriptase
Examples: human LINE and SINE elements, Drosophila copia elements
Simple insertion sequence (IS)
Class I
Transposable Element
Prokaryotic
Flanked on ends by small inverted repeats
Transposase gene encoded in internal sequence
Bacterial Insertion Sequences
Can harbor genes
Source of multiple drug resistance transfer
Eukaryotic Mobile Elements
Transposable Elements
Class I
Drosophila P elements
500 to 3000 base pairs
31 bp inverted terminal repeats
Encodes 2 proteins: Transposase and Repressor
Insertion into genes cause MUTATIONS
Transposase normally only expressed in germ line, where transposition can cause mutation
Repressor protein inhibits movement at other times
Drosophila P elements
used to generate mutants
Method: 2 lines crossed, one with P transposase, one with deleted P element unable to mobilize
Result: mobilized element produces insertional mutants in germ line of F1 flies
Maize Ac-Ds System in Maize
Another Class I system of transposable elements
Discovered by Barbara McClintock by mutation analysis in maize
20 years before discovery of bacterial transposons, 50+ years ago!
Led to her receiving a Nobel prize in 1983
Methods: analysis of maize mutations
Ds: Dissociation
Ac: Activator
Correlated genetic observations with cytological observations of maize chromosomes
Ac-Ds System Results:
Ds element located on chromosome 9
In absence of Ac, Ds is not transposable
In the presence of Ac, Ds can “jump” into genes, disrupting (mutating) them
Can also “jump” out of genes, restoring function
If Ac present in genome, Ds induced breakage adjacent to its location during somatic cell development
Chromosome breakage leads to phenotypic effects
Conclusions: What are Ac and Ds?
They are MOBILE GENETIC ELEMENTS
Ac contains two 11bp imperfect ITR
Ac contains 2 open reading frames (ORFs)
One of the ORFs encodes a transposase gene!
Ds is identical to Ac except for deletions in the transposase gene (there are a variety of Ds elements)…… this is what explains Ds dependence upon Ac for its transposition
Transposable elements in humans
Our genome is full of transposon DNA
Some estimates of up to one-half of our genome!
Examples: LINE (long insterpsersed elements); 6kb in length, up to 800,000+ copies SINE elements (short interspersed repeats), 100-500bases, ~1.5 million copies!
May move and cause mutation
Thought to have risen by reverse transcription of viral mRNAs followed by integration of dsDNA copies into the genome
No longer infectious due to deletions and mutations of viral genes
Makes up ~45% of human genome!!!
Movement of elements can be responsible for disease phenotype, eg. Alu SINE element in BRCA2 gene (familial breast cancer) or Alu in factor IX gene (hemophilia B)
Class II
Organisms
Human LINEs and SINEs, and copia (in Drosophila) are examples of Class II transposons: retrotransposons
Transpose through an RNA intermediate by Reverse Transcriptase polymerase that catalzyes creation of dsDNA from ssRNA
These retrotransposons resemble retroviruses in their sequence
May be the ancestral source of eukaryotic telomerase!
Maloney Murine Leukemia Virus
Transposable Elements Disease Example
Mutation Repair Mechanisms
Reversal of Damaged DNA
General Excision
Specific Excision
Post-replication Repair
Most repair processes rely on homology dependent repair mechanisms
-high fidelity:
excision repair
general
specific
-post-replication repair
Damaged DNA repaired by Photoreactivation repair:
E. coli prokaryotic repair mechanism
The bond creating the thymine dimer is cleaved by the photoreactivation enzyme (PRE), which must be activated by blue light.
Consequences of lack of nucleotide excision repair (NER)
Humans who lack the NER pathway
Unable to repair all damage due to UV radiation
Leads to xeroderma pigmentosum (XP)
Increased skin cancers and other skin abnormalities
Base excision repair (BER) is accomplished by uracil DNA glycosylase, AP endonuclease, DNA polymerase, and DNA ligase.
Uracil is recognized as a noncomplementary base, excised, and replaced with the complementary base (C).
Specific Excision
Through the process of recombination, the correct complementary sequence is recruited from the undamaged parental strand and inserted into the gap opposite the lesion.
The new gap created is filled by DNA polymerase and DNA ligase.
Recognition of newly synthesized strand is based on methylation
Post-replication Repair
Mutation Repair Systems Summary
Multiple cellular systems act to reverse or repair damage to DNA molecule
Fixed Mutations
Some mutations escape all repair mechanisms and become fixed
Synonymous mutations
Missense mutations
Nonsense mutations
Frameshift mutations
Consequences of Mutation
Genetic Variation that can be acted upon by natural selection (if mutations are fixed in germ line)
Somatic Disease Phenotype
Mutation —-> Disease
Large or multiple exposures to UV light leads to photodimers.
Repair mechanism is unable to repair all of the damage to DNA and some mutations become fixed.
Fixed mutations can lead to tumor phenotype (skin cancer).
Chapter 15 Summary
A low rate of spontaneous mutation occurs, “engine of evolution”
Many agents act to create DNA mutations, increase rate
Multiple repair mechanisms exist to repair mutations, decrease rate
Fixed mutations lead to altered gene product
——> altered phenotype
Kinase enzymes
- Modify other proteins by phosphorylating them (add a phosphate)
- The addition of the phosphate can change target protein’s localization, activity, or association with other proteins
- Kinases involved in cell signaling pathways and cell division pathways
- Mutated kinases associated with disease phenotypes including cancer
Kinase activation segment
domain frequently shown to harbor oncogenic mutations in known kinase cancer genes
P-loop
conserved structural region of kinases involved in ATP-binding
Kinase conserved regions
Kinase activation segment: domain frequently shown to harbor oncogenic mutations in known kinase cancer genes
P-loop: conserved structural region of kinases involved in ATP-binding
Observed mutations:
Mutational signatures differed between cancer types.
In the lung cancers, melanomas, and glioblastomas studied they may reflect exposure to tobacco carcinogens, UV light, and mutagenic alkylating chemotherapy, respectively.
Somatic mutations in cancer genomes
Driver mutations: confer growth advantage on the cell in which they occur, therefore positively selected for
Passenger mutations: not subject to selection, present in progenitor cell prior to clonal expansion, biologically neutral, do NOT offer growth advantage
Driver mutations
confer growth advantage on the cell in which they occur, therefore positively selected for
Passenger mutations
not subject to selection, present in progenitor cell prior to clonal expansion, biologically neutral, do NOT offer growth advantage
Approximately 120 of the 518 kinase genes screened are estimated to carry a “driver” mutation and therefore function as a “cancer” gene, more than previously anticipated
Analysis shows these some of these kinase mutations are associated with the FGF (Fibroblast Growth Factor) signaling pathway
Large scale screening of large numbers of somatic cancer tissues will provide more insight into cancer development and offer diagnostic and therapeutic opportunities (new sequencing machines capable of 300 billion bases per run will affect this!!)
Take home message:
The sequencing of the human genome now allows comparison with cancer genomes derived from malignant cells to determine location of somatic mutations and also to determine those that may be “driving” the malignant phenotype
