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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) DNA repair(2) meiotic recombination in all eukaryotes(3) sequestering of germ line in metazoans(4) 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
DNA polymerase III holoenzyme with subunits
(weight in daltons)
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
artificial template
double labeled probe
last nucleotide is non-complementary and labeled
non-complementary nucleotide excised, but no complementary
nucleotides
Replication
replication
A•Tketo
A•Tketo
template
daughter
A•Tketo
daughter
template
replication AT
replication AT
replication AT
replication AT
replication
A•Tenol
A•Tketo
template
daughter
G•Tenol
daughter
template
replication AT
replication AT
replication GC
replication AT
if unrepaired
Replication
replication
Aamino
•T
Aamino
•T
template
daughter
Aamino
•T
daughter
template
replication AT
replication AT
replication AT
replication AT
replication
Aimino
•T
Aimino
•C
template
daughter
Aamino •T
daughter
template
replication AT
replication GC
replication AT
replication AT
if unrepaired
Cytosine Tautomers : Camino•G Cimino•A binding
mutation from C to T
commonresults in
C•G pairing
rareresults in
C•A pairingAT substitution
Replication
replication
Camino
•G
Camino
•G
template
daughter
Camino
•G
daughter
template
replication CG
replication CG
replication CG
replication CG
replication
Cimino
•G
Cimino
•A
template
daughter
Camino •G
daughter
template
replication CG
replication TA
replication CG
replication CG
if unrepaired
Guanine Tautomers : Gketo•C Genol•T binding
mutation from G to A
commonresults in
G•C pairing
rareresults in
G•T pairing
Replication
replication
Gketo
•C
Gketo
•C
template
daughter
Gketo
•C
daughter
template
replication GC
replication GC
replication GC
replication GC
replication
Genol
•C
Genol
•T
template
daughter
Gketo
•C
daughter
template
replication GC
replication TA
replication GC
replication GC
if unrepaired
Mechanism of Frameshift Mutation: “Slipping a cog” …a base fails to pair with its partner during replication
Deaminationreplacement 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
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
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
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
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
problem: deletion of short nucleotide sequence
Repairing Double-stranded 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)
Undoing alkylation
Note that the enzyme is expended!
A tangible example of the importance of DNA repair