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Mutation and DNA Repair
Mutation Rates
Vary Depending on
Functional Constraints
Low Mutation Rates are Necessary for the Evolution of Complexity
1. Because most mutations are deleterious, there are limits to the number of mutations that an organism can
afford to accumulate in its somatic body, e.g.,
a) given mean eukaryotic rates, genomes can accommodate 60,000 genes without intolerable
mutational loads (Alberts et al.)
b) a mutation rate 10 times higher would limit genome size to ca. 6000 genes
2. Both the germ line and the somatic body must be protected from mutational load
(rare mutations become common because of large genomes and cell proliferation), e.g.,
a) germ line:
(1)
(2)
(3)
(4)
DNA repair
meiotic recombination in all eukaryotes
sequestering of germ line in metazoans
diplontic selection among cell lineages in meristems of plants
b) somatic tissues ...20% of deaths in western societies are due to cancer
(uncontrolled cell proliferation) resulting largely to the accumulation of genetic
damage in somatic tissues
(1) DNA repair
(2) immune systems
3. less efficient DNA repair and absence of meiosis may explain the limitation of prokaryotes
to small genomes and unicellular forms before the origin of these processes in the
protoeukaruote line.
4. spontaneous nucleotide changes are much higher than mutation rates would indicate, because of DNA
repair mechanisms
Two strategies to study gene function
• Genotype to Phenotype - sequencing
and searching for homologous
sequences, then study their function
• Phenotype to Genotype - mutational
screens and functional analysis
Kinds of Mutations
• Point Mutations
– Same sense mutations
– Missense Mutations
– Nonsense Mutations
– Transitions
– Transversions
• Frame shift mutations
• Substitutions, Deletions and Additions
Chemistry of single nucleotide substitutions:
a) transitions: a pyrimidine replaces a pyrimidine (C  T or T  C)
or a purine replaces a purine (A  G or G  A)
b) transversions: a pyrimidine replaces a purine or vice versa
c) transitions are less severe mutations that transversions:
(1) chemically, purines are more similar to one another than
they are to pyrmidines, and vice versa
(2) genetically, amino acid substitution is less likely with
transitions because of the degeneracy of the genetic code
(a) 3rd position transitions often code same amino acid
i) UUU and UUG both code for leucine
ii) GAA and GAG both code for glutamic acid
(b) 3rd position transversion less often codes for same
amino acid
i) UUU and UUG code for phenylanaline and leucine
Mutagenesis
• Spontaneous Mutations
– Replication Errors
– Other Errors
• Chemical Mutagenesis
• Radiation-induced Mutations
Replication Errors
Replication Proofreading
Mutator Strains of E. coli
• error prone replication
• mutD codes for e subunit of DNA pol III:
DNA polymerase III
holoenzyme with subunits
(weight in daltons)
e
Step 1: previous nucleotide pair is
tested for complementarity. If
passed, elongation occurs.
Step 2: If failed, the elongating strand is
transferred to the exonuclease site to
excise the mismatched nucleotide.
Experimental Demonstration of Proofreading
double
labeled
probe
artificial
template
non-complementary nucleotide
excised, but no complementary
nucleotides
last nucleotide is noncomplementary and labeled
Tautomerization of Bases
Thymine Tautomers: T•A to T•G binding
mutation from T to A
Replication
A
• replication
Tketo
A
• replication
Tenol
A
template
•
Tketo daughter
A
daughter
•
Tketo template
A
template
•
Tketo daughter
G daughter
•
Tenol template
replication
AT
replication
AT
replication
AT
replication
AT
replication
AT
replication
AT
if unrepaired
replication
replication
GC
AT
Adenine Tautomers : A•T to A•C binding
mutation from C to T
Replication
Aamino
• replication
T
Aimino
• replication
T
Aamino template
•
T
daughter
Aamino daughter
•
T
template
Aimino template
•
C
daughter
Aamino daughter
•
T
template
replication
AT
replication
AT
replication
AT
replication
AT
replication
AT
if unrepaired
replication
GC
replication
AT
replication
AT
Cytosine Tautomers :
Camino•G
Cimino•A binding
mutation from C to T
common
results in
C•G pairing
rare
results in
C•A pairing
AT substitution
Replication
Camino
• replication
G
Cimino
• replication
G
Camino template
•
G
daughter
Camino daughter
•
G
template
Cimino template
•
A
daughter
Camino daughter
•
G
template
replication
CG
replication
CG
replication
CG
replication
CG
replication
CG
if unrepaired
replication
TA
replication
CG
replication
CG
Guanine Tautomers :
Gketo•C
Genol•T binding
mutation from G to A
common
results in
G•C pairing
rare
results in
G•T pairing
Replication
Gketo
• replication
C
Genol
• replication
C
Gketo template
•
C
daughter
Gketo daughter
•
C
template
Genol template
•
T
daughter
Gketo daughter
•
C
template
replication
GC
replication
GC
replication
GC
replication
GC
replication
GC
if unrepaired
replication
TA
replication
GC
replication
GC
Frameshift Mutations
insertion
Mechanism of Frameshift Mutation:
“Slipping a cog” …a base fails to pair with its partner during replication
Spontaneous Mechanisms
Outside of Replication
Spontaneous hydrolysis can result in
deamination and depurination
Deamination
replacement of an amino group by a carbonyl oxygen
These nucleotide analogs have different
pairing affinities, but analogs can be
recognized and repaired
5-methyl C deamination results in T, which
can’t be recognized as a mutation
Replication produces a GC and an AT
C’s are selected for methylation in certain CG
sequences, which has led to the conversion
of most CG’s to TG’s during evolution
Deamination of C and A
illustrating different pairing behavior
Deamination and repair of C
Deamination
Repair of a
Deaminated
Cytosine
Deamination of
5-methylcytosine
Triplet Repeats
Pathology results when repeats exceed a threshold number.
Amplification of copy number by unequal crossing-over
Unequal crossing-over becomes more likely with increased copy number
Dynamic Mutations
Unequal crossing-over becomes more likely with increased copy number
and
The severity of the pathology increases with copy number
therefore...
Both the probability of the pathology and its severity increase over
generations after the number of repeats approaches the threshold
A number of conditions are based on this
mechanism operating in different genes
The repeats can be located in different orientations with
regard to the coding sequence
upstream
downstream
within
within
The repeats can be located in different orientations with
regard to the coding sequence ...even within a single gene
Chemical Mutagenesis
EMS is an alkylating agent
Nucleoside analogs can exhibit variant pairing behavior
keto (above); enol pairs to G instead of A
Acridine dyes intercalate DNA sequences
Effect: stabilizes the looping that leads
to deletions and insertions that cause
frame shift mutations
Mechanism of Frameshift Mutation:
“Slipping a cog” …a base fails to pair with its partner during replication
Major Repair Mechanisms
• Mismatch repair
• Excision repair
• Double strand breaks repaired mainly
by end-joining
• Inducible & error-prone mechanisms
Excision Repair
Excision repair
mechanism
More excision repair
modalities
Repair of UV damage
Thymine dimers
Excision Repair of UV
Induced Thymine Dimers
Mismatch Repair
Mismatch Repair
• To catch single-base errors that slip through proofreading
during replication
• Happens right after replication
• Misses C•C and small insertions and deletions
• mutH, mutL, mutS mutator strains are involved in
mismatch repair
• Trick is distinguishing the new daughter strand
MutH
Missmatch
Repair
How is the daughter
strand recognized as
the strand to correct?
GATC sequences methylated on
the 6 position of the A base
Mismatch
Repair
Mismatch
Repair
endonuclease
Activity
…nicks DNA
and then methylation
Radiation Induced Mutagenesis
• UV induced Thymine dimers
• Gamma and X-ray double stranded breaks
Spectrum
X-rays induce mutations
Multiple mechanisms to repair UV damage
Photo-activated
Repair System
Repairing Doublestranded Breaks
• often caused by radiation
(high energy gamma or X-rays,
directly or by creation of free
radicals)
• repaired by:
◊ Homologous recombination
◊ Blunt-end repair (right)
problem: deletion of short
nucleotide sequence
Inducible Repair
• Backup systems activated only in
emergencies
• Inducible
• Error prone
SOS
Undoing alkylation
Note that the enzyme is expended!
A tangible example of the importance
of DNA repair
Photo-activated
Repair System
Recombination
Repair
bulky mutations can leave
gapsafter replication
p53 activation of DNA repair