1. Xuhua Xia xxia@uottawa.ca http://dambe.bio.uottawa.ca
Mutation
Xuhua Xia

2. Outline Types of mutation Mutation rate and genome size
Mutation reveals the connection between genotype and phenotype; mutation and genetic diseases
DNA methylation and mutation

3. Mutations - any detectable change in DNA sequence
eg. errors in DNA replication/repair
inherited ones of interest in evolutionary studies
Deleterious
- reduce fitness and will be selected against by purifying selection)
Advantageous
increase fitness and have an increased chance of being fixed in the population by natural selection
Neutral
- will have little effect on phenotype
- may be fixed in population by genetic drift

4. Type of Mutations A G C T Point mutations transitions transversions
How many possible transitions? transversions?
p.38 “In animal nuclear DNA, ~ 60-70% of all point mutations
are TRANSITIONS, whereas if random expect 33%”

5. - different aa specified by codon
Missense mutation
- different aa specified by codon
Nonsense mutation
- change from sense codon to stop codon
Non-synonymous
- amino acid altered
Synonymous
- “silent” change (amino acid unaltered)
2. Insertions or deletions (“indels”)
- frameshift mutations within coding sequences
1 nt deletion
Fig. 1.12

6. Spontaneous Point Mutation Rates
G – genome size µb – mutation rate per site per generation (might have overestimated) µg – genomic mutation rate per generation
Table 4 in Drake et al. 1998, Genetics

7. Normal and Thalassemia HBb
----|----|----|----|----|----|----|----|----|----|----|----|--
Normal AUGGUGCACCUGACUCCUGAGGAGAAGUCUGCCGUUACUGCCCUGUGGGGCAAGGUGAACGU
Thalass. AUGGUGCACCUGACUCCUGAGGAGAAGUCUGCCGUUACUGCCCUGUGGGGCAAGGUGAACGU
**************************************************************
--|----|----|----|----|----|----|----|----|----|----|----|----
Normal GGAUGAAGUUGGUGGU-GAGGCCCUGGGCAGGUUGGUAUCAAGGUUACAAGACAGG......
Thalass. GGAUGAAGUUGGUGGUUGAGGCCCUGGGCAGGUUGGUAUCAAGGUUACAAGACAGG......
**************** ***************************************
Are the two genes homologous?
What evolutionary change can you infer from the alignment?
What is the consequence of the evolutionary change?
Xuhua Xia

8. Baccillus licheniformis Eschrichia coli 1st Baccillus subtilis
3rd
Baccillus licheniformis
Eschrichia coli
1st
Baccillus subtilis
Baccillus cereus
Mycoplasma capricolum
2nd
Thermus thermophilus
Streptomyces vanaceus
Anacyctis nidulans
Pseudomonas aeruginosa
Rhizobium parasponia
Micrococcus luteus
Muto A., Osawa S.: The guanine and cytocine content of genomic DNA and bacterial evolution. Proc Natl Acad Sci USA, 84(1987)

9. Do you agree or disagree with the following statement?
see p.27
“A synonymous mutation may not always be silent.”
GGT to GGA (3rd position change) within exon creates new splice site
Note: such mutations are detrimental and
relatively rare in nature !!
b-globin gene
(normal)
b thalassemia
disease
so part of globin coding sequence is missing in mRNA
& downstream exon is frameshifted
Evolutionary constraints on sequences near splice junctions:
- not only for amino acid encoded
- but also for cis elements required in splicing
Fig. 6.23
Goldsmith et al PNAS 80:

10. Lactase persistence (LP)
Segurel, L and Bon C Ann Rev 18:

11. Mutations leading to LP
-13910:C>T: Eurasia
-13907:C>G: East Africa
-13915:T>G: East Africa
-14009:T>G: East Africa
-14010:G>C: East Africa
LCT
Intron 13 of MCM6 (minichromosome maintenance complex component 6)
Segurel, L and Bon C Ann Rev 18:

12. Generation of short changes in length
Short insertions or deletions
(short “indels”)
eg. if slippage during DNA replication
- rapid evolution, change in copy number
of short tandem repeats
microsatellites
3’- TTAGGATCATATATATGTGCTTAA-5’
5’- AATCCTAGTATATATACACGAATT-3’
::::::::::::::::::::::::
Final outcome
Double-stranded DNA (Watson-Crick base-pairing) with 4 TA repeat units
Fig.1.18
Text figure also shows 2 TA repeat outcome (deletion)

13. wt: 10-26 CAG copies in tandem carrier: intermediate number
“Triplet repeat expansion” mutations
- increased copy number of tandem repeats of triplets within
gene (or regulatory region)
- certain human genetic (neurodegenerative) diseases
- repeat number strongly correlates with age of onset of disease
and severity
– copy number can change from one generation to next (“dynamic” mutations)
Huntington’s disease
htt gene on chr 4
Affected: copies
wt: CAG copies in tandem
carrier: intermediate number

14. Varying lengths of poly-Glutamine (Q) tracts (I & II) in androgen receptors in carnivores
Polymorphism among individual Amur tigers
N-terminus of protein I II …
Amur tiger
albino Amur tiger
Positions of aa identical to Chinese tiger protein are not shown.
- variation in poly(CAG) repeat length among species and within species
In humans normally: copies of CAG repeat
if higher #: correlation with muscular atrophy
if lower #: correlation with prostate cancer
giant panda
Use as biomarker
Wang Mol Biol Rep 39:2297, 2012

15. Triplet repeat disorder
- increased copy number of tandem repeats of triplets within
gene (or regulatory region)
- certain human genetic (neurodegenerative) diseases
- repeat number strongly correlates with age of onset of disease
and severity
DM1: Dystrophia myotonica-protein kinase, DMPK on Chr 19
DM2: ZNF9 gene on chromosome 3q21.
HTT on chr 4q16.3
Repeat copy number in normal = red;
orange = carrier;
yellow = disease condition
Gerald Karp Cell and Molecular Biology: Concepts and Experiments p.435

16. FMR1 protein/gene & Fragile X Syndrome
"fragile" is due to the methylation of FMR1 in Xq27.3 that is believed to result in constribution of the X chromosome which then appears "fragile" under microscope.
Bassell GJ, Warren ST Fragile X syndrome: loss of local mRNA regulation alters synaptic development and function. Neuron 60:
(Top) Protein domains (green) and key residues (red). NLS, nuclear localization signal; KH1 and KH2, RNA-binding domains; NES, nuclear export signal; RGG, RGG box, RNA binding. I304N, naturally occurring FXS mutation abrogating polysome association; S499, primary phosphorylated serine. (Middle) FMR1 gene, coding exons (blue) and untranslated regions (gray). Exons coding for major protein domains are indicated as well as alternative splicing. (Bottom) 5′ untranslated CGG-repeat alleles. The common and intermediate normal alleles (<55 repeats) are indicated, as are the premutation carrier alleles (55–200 repeats) and the full-mutation FXS alleles (>200 repeats).

17. Fragile X Syndrome: X-linked dominant

18. Huntington’s disease (autosome dominant)
4p16.3

19. 3. Inversions, translocations, etc.
through chromosome
breakage & rejoining
Inversion
NB: text shows as single-stranded, but both DNA strands take part in inversion event
Fig. 1.20
eg. if recombination
between indirect repeats
Region between them is inverted
Aside: inversions & translocations usually involve long DNA regions (eg. blocks of genes) rather than short stretches internal to genes

20. Outcome if recombination between direct repeats in genome?
A B
C D
A B
C D
A D
B C …
5’ ATTTCC 3’
5’ ATTTCC 3’
3’ TAAAGG 5’
3’ TAAAGG 5’
deletion of region located between direct repeats and generation of “extrachromosomal element” likely lost
(because no replication signals)
Fig. 1.17

21. Closer look at direct and indirect repeats…
Region “flipped over”
5’ ATTTCC 3’
5’ GGAAAT 3’
3’ TAAAGG 5’
3’ CCTTTA 5’
Fig. 1.20
C - G
U - A
A - U 5` 3`
Aside: At the RNA level , an inverted repeat can form a stem-loop structure

22. “Hot spots” of mutation
- short direct repeats, palindromes
- alternating Pu-Py dimers (Z-DNA)
- CpG in eukaryotes
-deamination of C to U,
repaired by uracil-DNA
glycolyase
- but 5-methyl C to T
escapes repair
patterns & positions of
mutations not random
Griffiths Fig. 7.16

23. Methylation and Evolution
Mutation and selection determines the pathway of evolution
Factors affecting mutation and selection will have evolutionary consequences
DNA methylation is interesting because it can modulate mutation and selection
H3C-
Methyltransferase
H3C
Donor Acceptor
Xuhua Xia

24. Different Methylation Processes
Methylation of amino acids in proteins
Methylation of nucleotide sequences (DNA and RNA)
Differences in methylation sites
Differences in functions
Illustrations
Methylation and DNA repair in Escherichia coli.
Methylation in the restriction-modification system
Methylation and gene regulation

25. Methylation and DNA Repair in E. coli
DNA alphabets: ACGT
RNA alphabets: ACGU
DNA duplication and Watson-Crick paring rule: A-T, C-G
H3C H3C H3C
3’--CTAG----CTAGGTAT----C-----C--CTAG ’
|||| |||||||| ? ? ||||
5’--GATC----GATCCATA----U-----T--GATC ’
mutH
mutS
mutL
H3C
Xuhua Xia

26. Methylation-Modification System
Bacterial
Genome
Restriction
enzyme
Methylase
TGGC*CA AC*CGGT
Transcription
and Translation
----TGG|CCA ACC|GGT---
Bacterial Membrane
dsDNA
phage
Brevibacterium albidum
Xuhua Xia

27. CpG-Specific DNA Methylation
Mammalian DNA methyltransferase 1 (DNMT1)
NLS-containing domain
replication foci-directing domain
ZnD, Zn-binding domain
polybromo domain
CatD, the catalytic domain
CpG mCpG mCpG
748
343
609
1110 1
RFDD
PBD
NlsD
ZnD
CatD
350
746
1620
613
1124
Fatemi, M., A. Hermann, S. Pradhan and A. Jeltsch, 2001 J Mol Biol 309:

28. CpG-Specific DNA Methylation
H3C
H3C
5’ATGCGA CCGA ACGGC--TAA 3’
|||||| |||| |||||
3’TACGCT GGCT TGCCG--ATT 5’
H3C
Fully methylated Hemi-methylated Unmethylated
Note: 5’CG3’ = CpG
Xuhua Xia

29. Methylation and Gene Regulation
Proteins with a methyl-CpG binding domain (MBD)
MBD1, MBD2, and MBD3
MeCP2
Histone deacetylases
Histone deacetylase
Condensed DNA with repressed transcription
MBD
---mCpG
Wade, P. A., and A. P. Wolffe, 2001 Nat Struct Biol 8:
Xuhua Xia

30. Methylation and Evolution
Mutation and selection determines the pathway of evolution
Factors affecting mutation and selection will have evolutionary consequences
DNA methylation, especially the CpG-specific methylation, can modulate mutation and selection
H3C-
Methyltransferase
H3C
Donor Acceptor
Xuhua Xia

31. Methylation and Mutation
NH2 N O
H3C
Spontaneous deamination
H3C
methylation N O
Cytocine is converted to Thymine
Xuhua Xia

32. CpG Dinucleotide Evolutionary consequence: CpG to TpG and CpA
CDS: 5’ATG CGA CCG AAC GGC …… TAA 3’
3’TAC GCT GGC TTG CCG …… ATT 5’
CDS: 5’ATG TGA CTG AAT GGC …… TAA 3’
3’TAC ACT GAC TTA CCG …… ATT 5’
Evolutionary consequence:
CpG to TpG and CpA
Decreased GC%
Xuhua Xia

33. CpG Deficiency in DNA
Variation in relative CpG abundance (RA) in prokaryotic genomes from 0.28 to 1.5
Variation in GC% in prokaryotic genomes from ~25% to ~75%
CDS: 5’ATG CGA CCG AAC GGC …… TAA 3’
3’TAC GCT GGC TTG CCG …… ATT 5’
CDS: 5’ATG TGA CTG AAT GGC …… TAA 3’
3’TAC ACT GAC TTA CCG …… ATT 5’
Xuhua Xia

34. Hypotheses on Variation in RA and GC%
Many hypotheses have been proposed to explain the variation in RA and GC%:
DNA methylation hypothesis (Bestor and Coxon 1993; Rideout et al. 1990; Sved and Bird 1990): Variation in DNA methylation causes variation in genomic CpG deficiency and GC%.
Stacking energy hypothesis with the prediction that all DNA sequences should have CpG deficiency (This is a vaguely specified hypothesis peculiarly with many advocates)
Constraint by amino acid usage (AA usage hypothesis) with the prediction that CpG deficiency should correlate with the frequency of amino acids coded by CpG-containing (i.e, CGN and NCG) codons.
Combination of AA and codon usage (AA and codon usage hypothesis): different genomes have different tRNA populations favoring C-ending codons differently. A genome favoring C-ending codons and containing many G-starting codons will be less CpG deficient than a genome not favoring C-ending codons.
Xuhua Xia

35. Problems
The methylation hypothesis has several major empirical difficulties (e.g., Cardon et al. 1994), especially in recent years with genome-based analysis (e.g., Goto et al. 2000):
Great variation in RA between Mycoplasma genitalium and M. pneumoniae.
Extreme CpG deficiency in Mycoplasma genitalium
Xuhua Xia

36. To Save the Methylation Hypothesis
The methylation hypothesis has to accommodate the arguments above:
How did M. genitalium get strong CpG deficiency without methylation?
Why there is so much variation between M. genitalium and M. pneumoniae without involving differential methylation activities?
Xuhua Xia

37. He who sees things from the very beginning has the most advantageous view of them.
-Aristotle

38. A New Hypothesis
Loss of methyltransferase gene
M. pneumoniae
M. genitalium
M. sp
Spiroplasma sp.
The reason for M. genitalium to have a strong CpG deficiency is because its ancestor had a methylation history.
Xuhua Xia

39. A New Hypothesis
Loss of methyltransferase gene
M. pneumoniae
M. genitalium
M. sp
Spiroplasma sp.
The reason for the variation in CpG deficiency between M. genitalium and M. pneumoniae is because of a much faster evolutionary rate in M. pneumoniae than in M. genitalium. Consequently, the former regains CpG much faster than the latter.
Xuhua Xia

40. Two Predictions and Tests
Mycoplasma sp. should be more CpG deficient than M. genitalium and M. pneumoniae.
M. pneumoniae evolves faster than M. genitalium.
Loss of methyltransferase gene
M. pneumoniae
M. genitalium
M. pulmonis
M. sp
Spiroplasma sp.

41. Results (1) Xuhua Xia Loss of methyltransferase gene
Methylation PCpG/(PCPG) GC%
Loss of methyltransferase gene
M. pulmonis
M. genitalium
M. pneumoniae
U. urealyticum
Xuhua Xia

42. Phylogenetic Analysis
Based on 18 sets of homologous CDS sequences with the 4 bacterial species
Two conclusions:
Mycoplasma pulmonis is closer to the root than the other two Mycoplasma species
M. pneumoniae have a longer branch length (i.e., the molecular clock ticks faster) than M. genitalium.
U. urealyticum
M. pulmonis
M. genitalium
M. pneumoniae
Xuhua Xia

43. Conclusions
Mycoplasma pulmonis, with several methyltransferase genes, is more CpG deficient than M. genitalium and M. pneumoniae.
M. pneumoniae evolves faster than M. genitalium.
The phylogenetic control and the biological significance beyond the conclusions.
Loss of methyltransferase gene
M. pneumoniae
M. genitalium
M. pulmonis
Spiroplasma sp.
Xuhua Xia