Lecture 21-22
15.5 Repairing Mistakes and DNA Damage
DNA polymerase III has an exonucleus active site, where an incorrect base pair moves to, catalyzes the removal of the incorrect deoxyribonucleotide
proofreading — ability of DNA polymerase to recognize and remove an incorrect deoxyribonucleotide
mismatch repair — error correction that cleans up errors from DNA synthesis
nucleotide excision repair — removes damaged DNA
16.4 Types and Consequences of Mutation
mutation — any permanent change in an organism’s DNA
Point Mutations
point mutation — alters the sequence of one or a small number of base pairs
missense mutations — change the identity of an amino acid in a protein
silent mutation — does not change amino acid sequence
frameshift mutation — shift the reading frame
addition/deletion of 1-2 base pairs
nonsense mutation — codon is changed to stop codon
3 categories
beneficial — helps organism’s fitness
neutral — silent mutations, does not affect fitness
deletorious — reduces fitness
Chromosome Mutations
deletion — broken segment of chromosome is lost
inversion — segments of broken chromosome may be flipped and rejoined
duplication — one or more additional copies of a segment
translocation — when a piece gets attached to a different chromosome
karyotype — complete set of chromosomes in a cell
Lecture Notes
replication, transcription, and translation all depend on accurate complementary base pairing, but none are perfect
dna replication is the most reliable — why?
mistakes in replication leads to alterations in the nucleotide sequence in the DNA
these alterations are passed on to daughter cells when the cell divides
heritable changes in the DNA are called MUTATIONS
in single-cell organisms, all daughter cells have the mutation
in multi-cell organisms, mutations can be somatic or germ-line
somatic (non-sex cell): passed to daughter cells in area
germ-line (sex cell): passed to new organisms
why are mistakes in transcription or translation not as critical?
mostly because of redundancy, as long as the blueprints are good
many copies of RNA and then protein produced
RNA’s and proteins are not heritable over multiple generations
important terminology: gene-, gene+
for example, lacI+ is the wild type (normal, unmutated)
lacI- is mutated form of the gene
DNA sequences can be changed by many factors:
uncorrected mistakes in replication
chemical mutagens
high-intensity radiation (x-rays, UV, etc)
among these, uncorrected mistakes in replication account for the most mutations
even if DNA replication is most reliable, it is not perfect
this is when millions to billions of new bonds are formed
how often do errors occur in DNA replication
frequency of mutations that end up in E. coli is about 0.04 mutations per cell division
can be represented as 4 mutations per 100 divisions
average in humans is about 1 mistake per billion nucleotides
but in vitro, E. coli DNA polymerases make ~400 mistakes per cell division — 10,000 times the observed number
so how do organisms reduce the frequency of the errors it makes by 10^4?
molecular “backspace” and “spell-check”
proofreading (a DNA polymerase’s “backspace key”): 3’-5’ exonuclease activity
if this exonuclease activity is gone, error frequency is 100 times higher (accounts for half of the 10000 difference)
if proofreading doesn’t catch the mistake:
mismatch repair system in E. coli scans recently synthesized DNA, looking for mismatches and hemimethylated (half-methylated) DNA
newly synthesized DNA is not methylated yet
new DNA gets methylated at adenine residues within the sequence 5’-GATC-3’ shortly after replication
once the daughter strands get methylated, there’s no way to tell which is the parent and which is the daughter, so the mismatch repair system doesn’t know which one is the wrong strand
mistake is in the new strand, so fix non-methylated strand
mismatch repair enzymes fix problem in unmethylated (new) strand
once DNA gets methylated, no way of distinguishing parent from daughter strand
methyl-directed mismatch repair (MMR) in E. coli:
three linked enzymes: MutS, MutH, MutL
MutS — scans for a mismatch
MutH — finds the hemimethylated DNA
MutL — links the two together, forms a bridge
MutH (endonuclease) cuts the nonmethylated (daughter) strand
an exonuclease removes the bases just before and just after the mismatch
DNA polymerase III fills in gap, ligase seals the nick
if mismatch repair enzymes are missing, the error frequency in DNA replication is 100 times higher
one type of colon cancer can be traced to a mutation in the human mismatch repair system
proofreading and mismatch repair each decrease the error frequency by 100x
100 × 100 = 10,000
what about mutations that arise form sources other than errors in DNA replication?
other specialized systems of repair enzymes recognize damage, and do their best to fix it
two broad categories of mutations:
point
base substitution (if in coding DNA regions):
same sense
missense
nonsense
frameshift
chromosomal-level
insertion
deletion
translocation
duplication
inversion
base substitution mutations
replace one base pair with another (ex. CG → TA):
transition: each base is replaced with a member of the same family (either purine or pyrimidine)
transversions: a purine becomes a pyrimidine and vice versa
how a base substitution becomes permanent:
the base-pair mismatch occurs during replication
one of the daughter strands of the parental strands replicates, forms a mutant with a changed base pair
three categories of base substitutions:
missense — codes for a different amino acid
nonsense — codes for a stop codon
same sense/silent — codes from the same amino acid
same sense mutations
a nucleotide (usually in the third position of codon) changed to a codon that specifies the same amino acid
no effect on protein product or function
what feature of the genetic code makes this possible? degeneracy, because more than one codon codes for the same amino acid
nonsense mutations:
codon specifying an amino acid is changed to a stop codon, causing premature termination
protein is truncated and virtually always inactive (null phenotype)
missense mutations:
codon specifying an amino acid is changed to a codon for a different amino acid; the effect on protein varies
if the amino acid that has been altered is not critical for the folding/function of that protein (ex. it’s not part of the alpha-helix or beta-sheets, it’s in between) or if the amino acid that replaces it assumes those duties in roughly the same way (have the same properties, ex. nonpolar, charged, etc.) then the protein is likely to retain some function, or even be fully functional
but if the altered amino acid is critical, and the duties aren’t taken up by the amino acid that replaces it then the protein will be inactive
ex: His/Arg vs His/Asp (positive/positive vs positive/negative) in active site, Glu/Asp vs Glu/Val (hydrophilic vs hydrophobic) in soluble protein
sickle-cell anemia
characterized by a defected beta-globin subunit in hemoglobin protein
single base pair (Glu → Val (hydrophobic)) substitution changes charge of protein
distorted cells get stuck in vessels, causing intense pain and anemia in low O2 conditions
normal hemoglobin do not associate with one another; each carries oxygen
sickle-cell hemoglobin because of the exposed hydrophobic Val crystallize together into a fiber, capacity to carry oxygen is reduced
1 in 600 African Americans have this disease
roughly 1 in 12 (8%) African Americans are carriers
in some tropical regions of Africa, around 45-50% are carriers
natural selection has not eliminated this disease due to the malaria defense it provides to carriers
phenylketonuria (PKU)
newborns are screened to prevent severe mental impairment; can be prevented by special diet
caused by single amino acid change in a 451 amino acid enzyme, phenylalanine hydroxylase
the mutated enzyme can’t convert phenylalanine to tyrosine, letting it build up, leads to mental impairment from the toxicity to nerves, and low levels of tyrosine leads to inhibited growth and inhibited melanin production
amino acid changes from base substitutions can be predicted with codon table:
changing last base — same box
change second base — same row
change first base — same column position
frameshift mutations
addition or removal of a small number of bases not divisible by three
disrupts the normal reading frame in the mRNA
all codons downstream of the change are altered
effect on protein product: usually results in premature appearance of stop codon → truncation of protien
in-frame removal of 3 bases
loss of one amino acid, rest of protein affected
doesn’t really count as a frameshift
cystic fibrosis is character by fault protein (CFTR)
70% of CF cases are due to an in-frame deletion of a Phe codon at AA 508 of a 1,400 AA protein
mutant protein doesn’t fold properly, never gets to cell membrane
Cl- accumulates in cells, causing an osmotic imbalance
mucus layer dehydrates, interference with breathing and digestion
chromosomal-level mutations
insertion/deletion
duplication
inversion
translocation (reciprocal)
some disorders caused by chromosomal-level mutations
some lymphomas and leukemias are caused by translocations
many oncogenes are activated and become cancers through duplications
fragile X syndrome and Huntington’s disease are also caused by duplications of short, repeated sequences
if inversions don’t involve the loss of DNA, they are often silent, by have been known to cause infertility
haemophilia A (loss of clotting factor VIII) is often caused by insertions
Duchenne’s Muscular Dystrophy is caused by a deletion
Ames Test for mutagenicity
bacteria cells
add chemical and rat liver enzymes to see what effect it has on the mutation rate