03/06 mutations Flashcards
what is ROS
oxidative radicals that can lead to oxidative damage when in overaccumulation
are ROS induced or spontaneous exclusively?
they occur naturally and spontaneously, but when under a certain mutagen, they can be induced into overaccumulation
how does ROS cause mutations
it changes the nucleotides which changes the base pairing and hydrogen bonding
if a mutation occurs by ROS and is not repaired before replication, would this affect gene expression
yes, it did not get repaired and is passed down
how does the body prevent overaccumulation of ROS
enzymes like superoxide dismutase and catalase
as well as antioxidants
if there is an increase in reactive oxygen species due to an environmental variable (high amounts of physiological stress or ingesting a toxin), then this would be considered an induced mutation
true
what is a trinucleotide repeat expansion (TRNE)
it is a sequence of 3 nucleotides that is increased from one generation to the next
what are some diseases affected by TNREs
huntingtons disease and fragile X syndrome
are TNREs spontaneous or induced
spontaneous
for a person with a TNRE disorder, the length of a trinucleotide repeat has:
increased above the critical size
if a TNRE occurs in a coding sequence, what type of expansion will occur
repeats of CAG
where can TNREs occur
within the coding sequence of a gene or in noncoding regions of genes
for a TNRE that occurs near a coding sequence, what typically occurs
a production of CpG islands
what is a CpG island
it results from a TNRE expansion that leads to the production of methylated cytosines and can lead to silencing and a reduction in gene expansion
the expansion of methylated cytosines near the promoter leads to?
silencing of the gene
what do TNRE sequences often result in
hairpins that form due to the complementarity of the repeat
if the TNRE hairpin forms before the DNA polymerase hits the sequence, what is the result
the DNA pol will slip over the hairpin and create a template that is too short
if the DNA polymerase slips off the template strand of a TNRE sequence and a hair pin forms, what is the result
an elongated sequence of the repeat
if the DNA polymerase slipped off the template at a TNRA sequence, what is the result of the repair enzymes
a expanded repeat or a repair back to its normal length
are TNRE repeat expansions always detrimental
no, they can have no affect depending on where they occur
what is a mutagen
it is a chemical or physical agent that alters DNA structure and induces mutation, it can be avoided
what are the three types of chemical mutagens
Base modifiers, intercalating agents, Base analogues
what are base modifiers? what type of mutagen are they
they are chemical mutagens
they are modifiers that covalently modify structures of nucleotides
what does the modification of bases by a mutagen do to the DNA
it alters the pairing in the daughter strand during DNA replication which results in mutation
mutagen modifications made to the template strand of DNA will result in:
inaccurate base pairing and mutations in the newly replicated strand
what are intercalating agents
intercalating agents contain flat planar structures that intercalate into the double helix
what is the effect of intercalating agents
distortion of helical structure
when DNA polymerase encounters intercalating agents in the template strand, what is the result
single nucleotide additions or deletions that result in frameshifts
which mutagens cause insertions and deletions
intercalating agents
if an intercalating agent such as ethidium bromide gets into a DNA strand, then it is likely to cause base substitutions
false, it causes insertions and deletions
what is a base analogue
it is a chemical mutagen that mimics bases and becomes incorporated into DNA
what is 5-bromouracil the analogue to?
thymine
in it’s normal, keto form, how will 5-bromouracil base pair? will this have any affect?
it will base pair with adenine, this will have no affect as it is acting normally
when 5-bromouracil tautomerizes, how will it base pair? will this have any affect on the DNA
it will mismatch and pair with G
this will cause mutation in DNA replication
what is a base analogue susceptible to?
it is VERY susceptible to tautomeric shifts
what are the two types of physical mutagens
ionizing radiation and nonionizing radiation
what is ionizing radiation
it is a very high energy form of radiation that is usually in the form of X Rays or Gamma Rays
what is the effect of ionizing radiation
it can deeply damage biological molecules and create free radicals
what is nonionizing radiation? how is it different from ionizing
it is lower forms of radiation usually in the form of UV, it does not include gamma rays and can be more easily blocked so it cannot penetrate deeply
what is a pyrimidine dimer
a pyrimidine dimer is a mutation caused by nonionzing radiation that causes a cross link between two pyrimidines on the same strand (next to eachother)
what is the impact of thymine dimers
they cause mutations when the DNA strand is replicated
what is the purpose of the Ames Test
test whether a suspected mutagen is in fact a mutagen
in the ames test, what mutation did the Salmonella already contain?
they were Hist negative, meaning that they cannot produce their own histidine
what is the difference between hist positive and hist negative for the Ames test
Hist positive refers to the wild type of salmonella, they can synthesize their own histidine
hist negative refers to the forward mutation that makes salmonella unable to synthesize their own histidine
in the ames test, what were they looking to measure
the rate of mutation for secondary (reversion) mutations that make the salmonella able to make histidine by themselves again
what was the control in the ames test?
Salmonella with a hist negative mutation and water.
These bacteria were placed on plates that did not have histidine on them
in the control, what was the purpose of the plates for the ames test?
the plates did NOT have histidine, so all bacteria that did not contain the reversion mutation would die
what type of bacteria can grow on the plates in the ames test?
only bacteria that underwent the secondary mutation to form histidine positive can grow on these plates
why did the plates in the control for the ames test show some bacteria when they should have had none?
spontaneous background mutations will always occur and some will result in the reversion mutation that brings the Hist negative back to positive
describe the experimental group set up of the Ames test
the hist negative salmonella were exposed to some suspected mutagen and plated on the same histidine absent plates as the control
if the suspected mutagen was actually a mutagen, what would the plates show in the Ames test?
They would show multiple colonies of growth above the control levels.
if the suspected mutagen was not a mutagen, what would the plates show in the Ames test
they would show a few bacteria on the plates that matched the control levels
what does it mean if multiple colonies above control levels grow on the histidine absent plates in the Ames test?
it means the suspected mutagen was actually mutacious and caused the secondary reversion mutation to histidine positive bacteria
what is the purpose of the Rat Liver Extract
detoxifying purposes
the rat extract makes the experiment more applicable to human conditions as it contains enzymes that can combat the mutagen
why were they looking at the rate of the mutation in the ames test
they wanted to see if the rate of mutation was increased above spontaneous levels when a mutagen was present
is the rat liver extract meant to cause more mutations?
no, it is meant to make the mutagen affects more similar to the human condition AND we are not interested in what the extract does to the DNA
We want to know what the extract does to the MUTAGEN itself
in the ames test, what is the initial state of the salmonella? what are we looking for with an introduction of a mutagen
the initial state is a forward mutation
we are looking for a reversion
what is the general process of DNA repair
1) surveillance proteins detect irregularity
2) repair enzymes remove abnormal DNA
3) normal DNA synthesized by DNA polymerases
what are the 6 types of repair mechanisms
direct repair
base excision
nucleotide excision
mismatch repair
homologous recombination
nonhomologous end joining
what is direct repair
a repair mechanism where a repair enzyme (not DNA pol) recognizes an error in DNA and converts it to the correct structure
which repair mechanism does not involve DNA polymerase
direct repair
what is the role of photolase in plants?
it is a form of direct repair that uses light to cleave dimers formed by nonionizing UV radiation
what is base excision repair
enzymes recognize abnormal bases and cleave the bond between the base and sugar
DNA polymerases act to repair
in DNA base excision repair, what is the first step?
N-glycosylase recognizes an abnormal base and cleaves the sugar and base bond
after the sugar-base bond has been cleaved in base excision repair, what recognizes the missing base and what occurs
an endonuclease will recognize it and form a nick on the 5’ side of the missing base
in base excision repair, what is the purpose of the nick?
it allows DNA polymerase’s 5’-3 exonuclease activity to remove the damaged region and fill it with new DNA
to fuse the bond between the newly repaired DNA and the phosphate backbone in base excision repair, what enzyme is involved?
DNA ligase
what is the primary difference between nucleotide excision repair and base excision repair
nucleotide excision removes and replaces large chunks of abnormal DNA
what are the similarities between nucleotide excision and base excision repair
they both require surveillance proteins to recognize errors
DNA polymerase fills in the new DNA once the error is removed
DNA ligase fuses them together
true or false
if we lose one of our repair mechanisms, we can still sufficiently repair mutations and be fine
false, you need all the repair mechanisms otherwise it can have detrimental consequences
what do mismatch repair systems generally do?
they detect and repair base pair mismatches
which strand is the mismatch system repair specific to? why?
it will digest the newly made strand because it is not methylated and is likely to contain the mismatch
why is methylation important to the mismatch repair system?
when DNA replication occurs, the new molecule is hemi-methylated. the parent strand is methylated while the newly made strand is not.
when replication occurs, the error is more likely to be in the new strand and since the new strand is not methylated, the repair system can identify which strand to digest
what type of proteins are involved in the identification of errors in mismatch repair
surveillance proteins remove the errors on the non-methylated strand
true or false
methyl directed mutation repair only works effectively when DNA has been recently synthesized since both of the strands of DNA will not have any methylation on it as of yet
false, it will be hemi-methylated
this is a requirement as this is how it identifies the errors and which to strand to digest
what are double stranded breaks
breakage of chromosomes into pieces
what are the mechanisms of repair for double stranded breaks
homologous recombination repair and nonhomologous repair
how does homologous recombination repair work
the sister chromatids are identical and can be used to repair one another by recombining and acting as a template for repair
when does homologous recombination repair work the most efficiently
it works best right after DNA replication when the sister chromatids are near eachother
what is non-homologous end joining? Between what chromosomes does this occur?
this occurs between chromosomes of different types
this is where two different chromosome pieces will get attached together as a last ditch means of repair
by what mechanism is the chromosome end of one transferred to another in a double-stranded break repair
translocation of non-homologous chromosomes
which DNA is repaired more efficiently? actively transcribed DNA or nontranscribed
actively transcribed is more susceptible to damage so it is repaired more efficiently
what is the transcription-repair coupling factor
it mediates transcription coupled with DNA repair
how does the transcription-repair coupling factor work
it targets actively transcribed genes with DNA damage and allows the RNA polymerase to come off the strand while the DNA is repaired before continuing transcription
what is the SOS response
it is where normal replication polymerases cannot replicate over damaged regions of DNA so it swaps with translesion polymerases that can handle the aberrant regions
what are translesion polymerases
they are polymerases that have flexible pockets that can accomodate aberrants structures in the template
what is the consequence of using a translesion polymerase
they have low fidelity and make errors
when 5-bromouracil undergoes a tautomeric shift, what type of base pair change will it cause
it will change a AT to a GC
