Minisatellites and microsatellites –
similar names but different biology
Yuri E Dubrova
Department of Genetics
University of Leicester
Tandem repeat DNA loci
¾ Consist of repeats
GAACAn
_GAACA_GAACA_GAACA_GAACA_GAACA ….
¾ Highly variable
up to 100+ alleles per locus
¾ High mutation rate
up to 0.15 per locus
¾ Because of this they are widely used in:
- forensics (paternity testing, identity identification)
- population genetics
- gene mapping
- mutagenesis (germline & somatic mutation induction)
- cancer studies (stability of cancer cells)
Tandem repeat DNA loci
¾Microsatellite loci
PubMed
26,529 items
¾Minisatellite loci
PubMed
3,120 items
¾Expanded Simple Tandem Repeat loci
(ESTR)
PubMed
63 items
Tandem repeat DNA loci
Repeat units
Homogeneous array
Heterogeneous array
Array
Minisatellites
¾ Repeat unit
¾ Size of array
¾ Complexity
of array
10 – 60 bp
0.5 – 15 kb
10 – 1,500 rpts
ESTRs
4 – 10 bp
0.1 – 20 kb
10 – 2,000 rpts
mostly
heterogeneous
homogeneous
Microsatellites
2 – 6 bp
10 – 1000 bp
5 – 200 rpts
mostly
homogeneous
Schematic examples of mutations at tandem repeat DNA loci
Gain of 2 repeats
X
Loss of 1 repeat
Germline mutations at human minisatellite loci
MS32
MS1
CEB25
paternal/gain
paternal/loss
maternal/gain
maternal/loss
Father
Child
Mother
Father
Child
Mother
Father
Child
Mother
Father
Child
Mother
MS31
Spontaneous germline mutation rates at
tandem repeat DNA loci and protein-coding genes
Mutation rate per gamete
Paternal Maternal
Total
Probe (Locus)
¾ Human minisatellite loci
9 CEB1 (D2S90)
0.161
0.003
0.082
9 B6.7 (20q13)
0.076
0.012
0.044
9 MS1 (D1S7)
0.055
0.049
0.052
9 CEB25 (D10S180)
0.035
0.019
0.027
9 CEB36 (D10S473)
0.018
0.018
0.018
9 MS31 (D7S21)
0.012
0.003
0.008
9 MS32 (D1S8)
0.009
0.006
0.007
¾ Mouse ESTR loci
9 Ms6-hm
0.10-0.18 0.06-0.09 0.09-0.13
9 Hm-2
0.02-0.07 0.04-0.14 0.04-0.11
¾ Human microsatellite loci mean ~ 0.002 (0.0001 – 0.01)
¾ Protein-coding genes
10-6 – 10-5 ( < 1 per 100,000)
Data from numerous original publications
Mechanisms of mutation
Microsatellites
Replication slippage
Predictions
¾ Mutate in dividing cells only
¾ No germline specificity, i.e.
mutations frequently occur
in somatic tissues
¾ Germline mutation rate
in males > females
¾ Age-related accumulation
of mutations
¾ Simple mutation spectrum
Cell divisions during spermatogenesis & oogenesis in humans
Spermatogenesis
Oogenesis
22 mitotic divisions for all ages
No male germ-cell divisions
Microsatellite mutation rates
Male/Female ratio 5 : 1
From: Crow, 2000, Nature Rev Genet, 1, 40-7; Ellegren, 2000, TIG 16, 551-8
Age, years
15
20
30
40
50
No replications
35
150
380
610
840
Spectrum of spontaneous microsatellite mutation in humans
(362 loci, 53 pedigrees, 630 subjects, 97 mutational events)
0.4
Frequency
0.3
0.2
0.1
0.0
1
2
3
4
5
6
7
No repeats lost or gained
From: Huang et al., 2002, Am J Hum Genet 70, 625-34
8
9
10
The big, the bad, the ugly microsatellites
¾ Long arrays are highly unstable, ‘100%’ mutation rate
¾ Associated with some human diseases
Secondary structures
Mutate in non-dividing tissues
DNA-repair is involved
(GAA)190 transgene in mice
Mutation frequency, %
20
16
2-month-old
12-month-old
12
8
4
0
Blood
Sperm
From: Clark et al., 2007, Hum Genet 120, 633
DRG
Cerebellum
From: Sinden, 1999, Am J Hum Genet 64, 346-53
Minisatellites
¾ Very unstable in the germline
but ~ dead in somatic tissues
¾ Very complex mutations in the male germline
and very simple in somatic tissues
Germline vs. somatic mutation at minisatellite loci
Sperm
Blood
Human minisatellite B6.7
CAG118 human microsatellite
transgene in mice
From: Tamaki et al., 1999, Hum Mol Genet 8, 879-88
Kovtun & McMurray, 2001, Nat Genet 27, 407-11
Spectrum of minisatellite mutants in sperm
From: Jobling et al., 2004, Human Evolutionary Genetics
Minisatellite mutation is somehow related to what is
going on at meiotic crossover hot-spots
Meiotic crossover
From: Jeffreys et al., 1999, Electrophoresis 20, 1665-75
Expanded Simple
Tandem Repeat loci
or ESTR
ESTR’s
¾ Repeat size:
4-10 bp, i.e. between micro- (1-4bp) & mini-
¾ Array size: 10-2000 rpts, i.e. more mini-like
¾ Unstable in somatic tissues, i.e. micro-like
ESTR mutation frequency, s.e.
0.12
0.10
Brain
Bone marrow
Sperm
0.08
0.06
0.04
0.02
0.00
BALB/c
F 1(C57 BLxCBA)
Similar spectra of spontaneous ESTR mutation in mouse tissues
Kruskal-Wallis Test
P = 0.7708
0.4
Brain
Bone marrow
Sperm
Frequency
0.3
But!
Intrastrand tetraplex
at the Ms6-hm locus
0.2
From: Weitzmann et al., 1998,
J Biol Chem 273, 30742-30749
0.1
0.0
3
6
8
12
16
No repeats lost or gained
20
30
60
If ESTR loci ‘behave’ like true microsatellites then:
¾ Age-related increases in replication-proficient tissues
¾ No age-related changes in non-dividing tissues
¾ Stage-specific pattern of spontaneous
ESTR mutation in the male germline
Age-related changes in ESTR mutation frequency in mice
ESTR mutation frequency, s.e.
0.4
0.3
Sperm
0.2
Bone marrow
0.1
Brain
0.0
12
26
48
Age, week
96
A single Sertoli cell with its associated germ cells
From: Cooke & Saunders, 2002, Nat Rev Genet 3, 790
Meiotic
spermatocytes I
Round
spermatids
Elongated
spermatids
Meiotic events
Pre-meiotic
spermatogonia
Late post-meiotic events
Pre-meiotic
Meiotic
spermatogonia spermatocytes I
Elongated
spermatids
Round
spermatids
Mitotic events
Pre-meiotic
Meiotic
spermatogonia spermatocytes I
Round
spermatids
Elongated
spermatids
Separation of mouse germ cells by flow cytometry
Spermatocytes I, 4C
80-100,000 cells
Spermatogonia/stem cells
2C, 50-80,000
Elongated spermatids
1C, 100-150,000 cells
Round spermatids
1C, 150-230,000 cells
0.00
Elogated s'tids
Round s'tids
Male 2
Spermatocytes
Spermatogonia
Elogated s'tids
Round s'tids
Male 1
Spermatocytes
Spermatogonia
0.25
Elogated s'tids
Round s'tids
0.30
Spermatocytes
Spermatogonia
ESTR mutation frequency, 95% CI
ESTR mutation frequencies in mouse germ cells
Male 3
ESTR mutation events
are attributed to mitosis
0.20
0.15
0.10
0.05
Problems
We observe changes in mutation frequencies, p
p = f(u, N, s)
u – mutation rate per DNA replication/cell division
N – number of cell divisions
known for the male germ cells
s – selection against mutants, s = 0
?
Mutation induction at mouse ESTR loci
Paternal mutation rate, 95% CI
0.5
Fission neutrons
0.4
Chronic γ-rays
1.66x10-4 Gy/min
0.3
0.2
Acute X-rays
0.5 Gy/min
0.1
0.0
0
0.25
0.5
Dose, Gy
From: Dubrova et al., 1998, PNAS 95, 6251-6255
2000, Mutat Res 453, 17-24
0.75
1
Mutation induction at mouse ESTR loci
ENU
iPMS
ESTR mutation rate, 95% CI
0.25
0.20
0.15
0.10
0.05
0.00
0
12.5
25
50
75
Dose, mg/kg
From: Vilariño-Güell et al., 2003, Mutat Res 526, 63-73
0
12.5
25
37.5
Non-targeted mutation induction at mouse ESTR loci
Expected genome damage from mouse ESTR data
¾ Spontaneous mutation rate (Ms6-hm + Hm-2) 0.055 per locus
¾ Induced paternal mutation rate (1 Gy)
0.225 per locus
¾ Radiation-induced increase in mutation rate
0.170 per locus
¾ Mean size for Ms6-hm & Hm-2 (CBA/H mice) 5 x 103 bp
¾ Genome size
3 x 109 bp
¾ Damage to the whole genome
0.17x(3x109)/(5x103)=100,000
Radiation-Induced Damage in Eukaryotic Cells
¾ Base damage
¾ Single-strand breaks
¾ DNA-protein links
¾ Double-strand breaks
¾ Bulky lesions
¾ Total
From: Dubrova et al., 1998, PNAS 95, 6251-6255
Frankenberg-Schwager, 1990, Radiat Environ Biophys 29, 273-292
2000 per 1Gy
1000 per 1Gy
150 per 1Gy
40 per 1Gy
40 per 1Gy
3300 per 1Gy<<100,000
ESTR mutation induction and cell division
Stage of irradiation
Tissue, proliferation capacity
Brain
Bone marrow Male germ
cells
Embryo males
12 days of gestation
+
+
+
-
-
-
-
+/-
+
Adult males
1 week after exposure
Adult males
10 weeks after exposure
ESTR mutation induction in male mice exposed to 1 Gy X-rays
0.14
P=0.0002
P=0.0004
P=0.0011
P=0.0066
0.10
0.08
P=0.0619
0.06
0.04
In utero
8 weeks
1 week
Sperm
In utero
8 weeks
BM
In utero
8 weeks
1 week
0.00
1 week
ESTR mutation induction occurs
in replication-proficient tissues
0.02
Brain
ESTR mutation frequency, s.e.
0.12
0 Gy
1 Gy
Problems
¾ We observe changes in mutation frequency
Mutation rate
¾ The magnitude of changes in mutation rate per cell
division which corresponds to the observed increase