lecture 11 & 12- DNA mutations Flashcards
a mutation is a change in ___ that is propagated through ___
DNA sequence
cellular generations (has to be fixed mutation)
when mutations occur in germ-line cells, they are ___
inheritable
how can mutations be good
evolution
- some frequency of mutation is necessary to produce variability in which natural selection acts to drive evolution
when does a DNA alteration become a stable, inheritable mutation?
only after the alternation is converted through replication into an incorrect base pair (such as an A-T pair where a G-C should be)
what happens to the vast majority of damaged nucleotides that occur in mammalian cells?
fixed by DNA repair enzymes
- they can fix the alterations in DNA sequence, but if not fixed by next round of replication –> passed down (inheritable)
point mutations are ___
single base pair substitutions
transitions exchange a ___ for a ___
transversions exchange a ___ for a ___
purine for a purine (A–>G)
pyrimidine for a pyrimidine ( T–>C)
purine for a pyrimidine (A–>C)
pyrimidine for a purine (T–>G)
name the two types of point mutations
transitions
transversions
mutations of one or a few base pairs usually result from what?
errors in replication or damaged nucleotides
what is more common- transitions or transversions?
transitions are 10 times more common
a point mutation in the protein-coding region of a gene can result in what?
these point mutations can be classified as…
can result in an altered protein with partial or complete loss of function
- silent, missense, nonsense
describe RNA complementarity to the DNA strands
RNA has same sequence as DNA top strand (coding- sense, nontemplate strand)
RNA is complementary to DNA bottom strand (noncoding, antisense, template strand)
describe genetic code codons
DNA sequence encoding a protein is read in triplets (64 codons)
- 61 of 64 codons are sense codons for AA’s
- 3 of the 64 are nonsense- stop codons
UAA, UAG, UGA
- start codon = AUG (Met)
- code is degenerate (a single AA can be encoded by more than one codon)
. exception = Met (only encoded by AUG)
name 2 rules in genetic code
1- no overlapping in triplets, read from a fixed point
. if it wasn’t, it would code different AA’s
2- open reading frame- a run of sense codons before a stop codon is encounters
describe silent mutations
- mutations in non-coding or non-regulatory regions of DNA
- in coding regions- b/c of degeneracy, results in same AA
- ex: change from TTT to TTC is silent because both code for lysine (they are synonymous)
describe missense mutations
any mutation within coding region leading to change in amino acid
- protein could have loss of function or gain of function if sequence change is in active site- severity depends on characteristics of the AA replaced (depends on nature and location of the change)
- ex: sickle-cell anemia- caused by missense mutation in gene that codes for beta subunit of hemoglobin - beta subunit has different shape, leads to aggregation of protein when with alpha subunit (change is glutamic acid –> valine)
describe nonsense mutation
leads to stop codon, terminating translation
- generates a truncated protein without a complete AA sequence
- severity depends on location
establishment of point mutations is a 2 step process…
1- create mutation- DNA polymerase incorporates an incorrect nucleotide
2- if mismatch is not repaired, it becomes a mutation in SECOND ROUND of replication
insertions occur when…
deletions occur when…
insertions occur when one or more base pairs are added to the wild-type sequence
deletions occur with loss of one or more base pairs
insertions and deletions may have no effect if…
not in genes or regulatory sequences
an indel mutation of only one or two base pairs in the coding sequence of a protein throws off the reading frame after the mutation, resulting in a ___
frameshift mutation
indels of 3 or multiples of nucleotides do what?
preserve the reading frame of the gene
- most common: insertion of 3 nucleotides (template slippage by DNA poly during replication) –> triplet expansion diseases, often CAG repeated triplet
name 4 DNA alterations that lead to mutations
1- spontaneous hydrolysis
2- oxidative damage (ROS)
3- alkylation (alkylating agents)
4- radiation (UV, x-rays)
spontaneous DNA damage by water can cause point mutations by ___ or ___
deamination of nitrogenous bases (C, G, or A) or loss of bases by hydrolysis
deamination is the removal of a ___ group and changes ___
amino
changes identity of bases and their pairing properties
describe deamination with cytosine
deamination of cytosine creates uracil
however, about 5% of cytosines in higher eukaryotes are methylated
- deamination of methylcytosine leads to thymine
describe deamination with guanine or adenine
less frequent and produces abnormal bases
- guanine becomes xanthine, pairs with cytosine
- adenine becomes hypoxanthine, pairs with cytosine
describe DNA damage by spontaneous hydrolysis
loss of bases by hydrolysis
- depurination much more frequent than depyrimidation
- 1 in 10,000 purines are lost from mammalian cell DNA every 24 hrs
oxidative damage and alkylating agents can create ___ and ___
point mutations and strand breaks
describe oxidative damage
sodium nitrate (common food preservative) and nitrosamines are converted to nitrous acid in the stomach (nitrous acid is potent mutagen)
- oxidative damage is possibly the most important source of mutagenic alterations in DNA
- ROS, H2O2, hydroxyl radicals (OH-) and superoxide radicals (O2-) arise during irradiation or as byproducts of aerobic metabolism
- oxdative damage includes modification of bases, sugar, removal of bases and strand breaks
what is possibly the most important source of mutagenic alterations in DNA
oxidative damage
oxidative damage includes…
modification of bases, sugar, removal of bases, and strand breaks
describe oxidative guanine
oxidative guanine at position 8 is very mutagenic because it can pair with adenine, leading to a transversion if not repaired prior to replication
. instead of G-C, T-A –> one of the most common mutations in human cancers
describe damage by alkylation
addition of an alkyl group (methyl, ethyl, propyl, isopropyl) to bases
describe how guanine is damaged by alkylation
O6-methylguanine is very mutagenic because it tends to pair with T instead of C, leading to a transition (G–>A)
give two examples of damage by alkylation
1- benzo pyrene (carcinogen, intercalating agent)- smoke of burning cigs, woodm coal
. in liver, becomes a reactive epoxide that can react with bases, especially guanine
2- nitrogen mustard gas- cross-linking agents that react with adjacent guanine residues, preventing replication and transcription
how are DNA-damaging agents used in chemotherapy?
DNA-reactive agents used in chemo for cancer kill cells (cytotoxic) by creating broken chromosomes or stalled replication forks, both lead to cell death during cell division
- cross-linking agents
- intercalating agents- doxorubicin- stops replication forks, cells can’t divide anymore
what is the Ames test used to identify
DNA-damaging, mutagenic chemicals in animals
describe how the Ames test works
use histidine auxotrophic Salmonella typhimurium- mutation in biosynthetic pathway for histidine (requires histidine in growth medium)
- looks for his revertants
- when his- bacteria (S. typh) is plated in media without histidine and chemical-potential mutagen is added, cells show a reversion mutation (can synthesize his and grow in his-free medium) (the chemical led to mutations)
most known human carcinogens result in what in the ames test?
increased mutation in the Ames test
- compounds identified as mutagens in an ames test require further testing to determine whether they are likely to be human carcinogens
describe DNA damage caused by radiation
ionizing radiation: UV (in sunlight), cosmic rays, x-rays
- UV and other ionizing radiation: about 10% of all DNA damage caused by environmental agents
- UV can cause photochemical fusion of adjacent pyrimidines (pyrimidine dimers- covalent cross-links) – leads to no replication
- x-rays and gamma rays (higher energy): single or double-stranded DNA breaks
UV light from the sun can cause ___ that stall ___
pyrimidine dimers that stall DNA polymerase during replication
x rays and gamma rays cause ___
single and double stranded DNA breaks
DNA-damaging agents can lead to mutations at ___ concentrations and can kill the cell at ___ concentrations
low
high
