Lecture 3/17 Flashcards
If the bar eye is X-linked, is a male more likely to inherit it if it’s a recessive or dominant gene?
it doesn’t matter, as it will only get one copy. if the copy it gets is the bar, it will have bar eyes
are mutagens only seen in the environment?
no, they are all actually endogenous (naturally occurring in the body) as well as environmental
which is worse – increased ROSs or DSBs?
double stranded breaks are worse
what causes a DSB?
X-rays, UV boxes, radiation treatment
what is an intercalating agent? how is it a mutagen?
something that inserts itself between bases, causing a disruption in DNA replication/repair/recombination –> it makes the DNA think there is a mismatch, which leads to the deletion of a base pair or insertion of a random base pair
what are the different types of mutagens?
intercalating agents, base analogs
what are base analogs? what’s an example and how does it work
molecules that mimic the structure of a standard base. example is uracil with bromide. uracil with bromide-rare state behaves like cytosine, so it pairs to guanine
what are the effects of a somatic mutation?
- non germ cells
- not transmitted to next generation
- can affect survival of an individual
- can lead to cancer, but doesn’t affect progeny
what are the effects of germ line mutations?
- occur in gametes/gamete precursor cells
- doesn’t affect the parent with the mutation, only future generation
- transmitted to next generation
- provides raw material for natural selection
- these mutations are often cancerous
what can cause a mutation to not be permanent?
if the DNA repair occurs before replication
what is the effect of a mutation dependent upon?
if the mutation is corrected and if it’s in a somatic cell or germline
what helps DNA be repaired before replication
having a vigilant repair system
at what point are mutations usually corrected? does it do a good job? how does it work?
during proofreading, it’s highly accurate. mispaired base is recognized and excised by 3’ to 5’ exonuclease activity of DNA pol; improves fidelity of replication 100-fold
what is alkyltransferase?
it removes alkyl groups (alkyl transferases are naturally occurring)
how do prokaryotes respond to UV ray-caused thymine dimers?
photolyase is used to split the covalent bond of thymine dimers in presence of light
which kinds of repair are single strand base repair?
homology-dependent repair:
- base excision repair
- nucleotide excision repair
which kinds of repair are for double stranded breaks?
correction of DNA replication errors:
- methyl-directed mismatch repair
how does base excision repair work? (5steps)
- deaminated C DNA looks like a U, glycosylase recognizes and cleaves the C from the sugar, leaving an AP site.
- AP endonuclease cuts in and nicks the backbone at the AP site around where the mutation occurred
- DNA exonucleases remove nucleotides near the nick because there’s a free 3’ and 5’ end (which is bad)
- DNA pol I/beta synthesizes new DNA to fill the gap
- DNA ligase seals the nick
what is an AP site?
an apurinic or apyrimidic site
does AP endonuclease make a ss or ds cut?
single stranded
what do glycosylases do?
recognize different types of DNA damage and cleave them
what happens if you have mutations in/are missing glycosylases?
you die, these are essential enzymes
what type of repair uses glycosylases?
base excision repair (ber)
what happens in response to UV exposure?
thymine dimers
what are the steps of nucleotide excision repair (ner), 7 steps?
- exposure to UV –> thymine dimer
- UvrA in complex with UvrB recognize DNA distortion region. Once the legion is found, UvrB binds and UvrA dissociates.
- UvrB now forms a complex with UvrC, which has 2 endonuclease pockets to nick the DNA 5’ and 3’ to the thymine dimer
- damaged ss fragment is released from DNA by UvrD (helicase)
- DNA pol I/k/d/e uses the 3’ end to fill in the gap with new DNA and ligase reseals the repaired strand
what is the difference between ber and ner?
Base Excision Repair (BER) repairs small, non-helix-distorting base lesions, while Nucleotide Excision Repair (NER) addresses bulky, helix-distorting DNA damage, like those caused by UV light
which active agent in UV damage repair acts as helicase?
UvrD
how does methyl-directed mismatch repair work?
a methyl group is added to A in GATC sequence and the newly replicated DNA isn’t yet methylated.
1. MutS recognizes mismatches not caught by proofreading.
2. MutS with MutL (in a complex) recruit and activate MutH, which has endonuclease activity. MutH nicks the unmethylated strand opposite the nearest methylated GATC
3. DNA exonucleases make a gap in the unmethylated strand by excising DNA.
4. Gap filled in by DNA pol iii using the methylated strand as template; the fragments are ligated. New strand later gets methylated.
where does methyl-directed mismatch repair occur?
in bacteria
in bacteria, which strand is marked by adenine methylase?
parental strand
is it always the same DNA exonuclease recruited for methyl-directed mismatch repair?
no, it depends on where the methylation site is
where does MutH cleave?
near a methylated site, but how much it cleaves depends on how close it was to the actual site – it can be 3’ or 5’ to the actual mistake
how does methyl-directed mismatch repair work in euks?
- methylization is not used to distinguish parental vs. newly synth.d strand
- same as in bacteria, but the repair enzymes are MutSalpha/MutSbeta and MutLalpha/MutLbeta along with other proteins; MutL proteins have endonuclease activity; MutH recognizes parental vs new strand
do we know how strands are distinguished for methyl-directed mismatch in euks?
no
mutations in MutSalpha and MutLalpha increase rates of what?
colorectal cancer
what error prone repair mechanisms are in bacteria?
the SOS system
how does the SOS system work?
- it is used at replication forks that stalled bc of unrepaired DNA damage, “sloppy” DNA pol is used instead of normal polymerase
- adds random nucleotides opposite damaged bases
what types of repair mechanisms are error prone in euks?
repairs for double stranded breaks:
- nonhomologous end-joining (NHEJ)
- homologous recombination
how does non-homologous end-joining work?
- DSBs recognized by PK/KU80/70 complex, it comes in immediately and binds to the free end
- the complex recruits other proteins for protection, to hold the broken structure together so it can be repaired with itself and not get ligated to something else, and end-trimming of DNA
- ligation of ends (this can sometimes occur between fragments of different chromosomes, as many DSBs can happen concurrently)
are double stranded breaks clean breaks?
they can be, or they can have overhang.
what is end trimming?
it is resection and can lead to loss of DNA, it sometimes gets rid of more than just overhang
what is one downside to ligation of ends in DSB repair?
if ligation occurs between fragments of different chromosomes by accident, it can lead to lethal chromosome rearrangements (ie. deletions, inversions, translocations)
how does homologous recombination work for DSB repairs?
- MRN complex (Mre11, Rad50, Nbs1) competes with KU complex to recognize DSB.
- resection of 5’ end (CtIP complex) –> similar to recombination
- Rad51/BRCA binds to 3’ ends with help of other proteins
- searches for homologous regions for 3’ tail invasion. DNA ligase
when does homologous recombination vs. nonhomologous end-joining occur for DSB repair? which would be better to occur?
it depends, as the MRN complex and Ku complex compete to recognize and bind to the DSB? better for homologous recombination to occur
which type of DNA repair mechanism would be lethal with a mutation in the mechanism?
base excision repair
what can a mutation in ner cause?
many diseases, all resulting in individuals particularly sensitive to light bc of UV
what do mutations in helicase used in DNA repair cause?
diseases that cause premature aging
what does a mutation in dsb repair cause?
breast cancer/prostate cancer, ataxia telangiectasia (loss of coordination and dilated capillaries)
how does the control test of the Ames test work?
control test:
two tubes, one with his(-) mutant bacteria and one with rat liver enzymes; mixture is plated onto medium without histidine and you expect no growth. any growth shows his(+) revertants
how do you calculate rate of spontaneous mutations
number of mutations/number of plated cells
how does the mutagenicity test of the Ames test work?
three tubes, one with his(-) mutant bacteria and one with rat liver enzymes, one with potential mutagen; mixture is plated onto medium without histidine, any growth shows his(+) revertants
what are the two tests required by the FDA to test potential carcinogens?
1) bacterial reversion test
2) mammalian cells in vitro or in vivo (measure of # of chromosomal aberrations in metaphase chromosomes)
what level above spontaneous mutations does the FDA approve for products to be used on humans?
nothing above spontaneous mutation level
is benzo[a]pyrene a carcinogen?
yes, but only once it has been processed in the liver
