DNA repair in plants studied by comet assay
Karel J. Angelis, Jaroslav Kozák*, Radka Vágnerová and Marcela Holá
Institute of Experimental Botany AS CR, Czech Republic
*Institute of Organic Chemistry and Biochemistry AS CR, Czech Republic
2015 ICAW Antverpen
1) Introduction: Why SNGE
2) Comet assay protocols-principles and sensitivity
3) Model plants, tissues and age stages
4) Induction of DNA damage
5) Repair of SSBs
6) Repair of DSBs
7) Detection of mutations in PpAPT locus
8) Conclusions
Plant comets: nuclei or protoplasts?
Physcomitrella comets prepared from isolated nuclei and protoplasts
nuclei from 1 week old protonema
Comet from protoplasts prepared 2
hour after removal from Dricellase
Preparation of nuclei from plant tissue
2 minutes from start of chopping the tissue to dipping
comet slide into lysis buffer
Shown frozen Arabidopsis seedlings, nevertheless chopping with razor blade
could be applied virtually to any frozen plant tissue
Comet assay
pH of lysis and electrophoresis determine protocol:
N/N
(neutral lysis and electrophoresis) protocol – DSB
A/N
(alkali unwinding/neutral electrophoresis) protocol – SSB
A/A
(electrophoresis in 0.3M NaOH) protocol – radical sensitive,
rather than alkali labile sites
Sensitivity of comet protocols to detect X-ray induced damage
Vicia faba root tips
X-ray dose rate 1 Gy/min
A/A – protocol: 0.3 M NaOH
A/N – protocol: TBE after 0.3 M NaOH
N/N – protocol: TBE
Arabisopsis thaliana
Root culture –
differentiated tissue
Seedlings (10 days old)
mix of dividing meristem
and differentiated cells
Suspension culture –
dividing tissue
The moss Physcomitrella patens
The life cycle of Physcomitrella:
(1) Spores, (2) germinating spores generating primary chloronemata, (3) 15-d-old protonemal colony, (4) branching
chloronema, (5) caulonema cells, (6) filamentous structure switches to three dimensions with young buds, (7) bud
developing to leafy shoot - gametophore, (8) a fully developed gametophyte (4 weeks old), (9) an archegonium
(female structure), (10) two antheridia (male structures), (11) after fertilization the egg cell develops into a small
diploid sporophyte and within its capsule meiosis occurs leading to spore formation. The whole cycle can be
achieved under optimal conditions in less than 12 weeks. Plant Physiol. 2001.
Physcomitrella protonemata for comet assay
Stages after 1, 7 and 14 days after passage
1 day
7 days
14 days
4-7 cells per filament
(30-50% apical cells),
dividing tissue
7 days app. 20 (~ 10% apical) cells per filament, growing tissue
14 days > 20 (< 5% apical) cells, differentiated tissue
Bleomycin/ionizing radiation induction of DNA breaks
massive DNA attack generally by oxygen radicals
Induction of DSBs and SSBs by Bleomycin treatment
100
A/N
N/N
% DSB remaining
80
60
40
A/N protocol
20
0
N/N protocol
0
10
20
30
mg/ml Bleomycin
40
50
Arabidopsis and Physcomitrella sensitivity to MMS
- Arabidopsis roots (black)
100
- seedlings (green)
80
%T DNA
At roots
60
At seedlings
40
At suspension
- suspension culture (red)
- Physcomitrella 1 day (blue)
Pp protonema
20
0
0
2
4
6
mM MMS
8
10
1 hour treatment
Repair (BER) of SSBs induced by MMS in Arabidopsis
140
120
SSB remaining (%)
Col0
100
atparp1
80
60
Col0 +
AG14361
40
Col0 +
3ABA
20
0
0
60
120
180
240
300
360
t-minutes repair
- col0 SSB repair half time ~ 6h
- atlig1 > 9 h
- AtLIGI in plants substitutes
DNA ligase III in BER
Waterworth et al. 2009
Poly (ADP-ribose) polymerase (PARP) is a family of
proteins also involved in DNA repair.
- AG14361 - selective inhibitor of PARP1
- 3ABA – broad PARP inhibitor
Kinetics of UV induced NER SSBs incisions and CPDs in
Physcomitrella
UVC irradiation 1kJ
% damage remaining
150
DNA ssb
100
CPD detection:
homemade T4EndoV
dillution 1:1000
digestion 30 min RT in
50 mM Tris, 5 mM EDTA, pH 7.5
CPDs by
T4EndoV
50
0
0
1
2
3
t-hours
4
5
6
Holá et al. 2015
Repair kinetics of DSBs in Arabidopsis
100
atmim
(smc6b)
atlig1
% DSB remaining
80
atrad21.1
60
wt
40
atlig4
atku80
20
0
0
20
40
t (minutes)
60
Parameters of DSB repair kinetics
mutant
tissue t1/2 fast % fast t1/2 slow
At wild-type
atku80
atlig4
atmim
atlig1
atrad21.1
10 day seedlings
Two phase decay analysis
kinetics
4.2
50.2
10.8
two phase
1.4
59.3
9.8
two phase
1.3
49.8
13.4
two phase
75.3
one phase
14.6
one phase
11
one phase
does not fit
two phase
kinetics
Kozák et al, 2009
Physcomitrella mutants defective in DSB repair
Bleomycin treated 1 hour of 1 d protonema, plated and grown for 10 days
wt
BLM ng/ml
0
10
100
1000
mre11
nbs1
rad51AB
rad50
Repair kinetics of Bleomycin induced breaks in wt Physcomitrella
SSB – A/N protocol
DSB – N/N protocol
Treated protonemata:
1, 7 and 14 days old
Repair of Bleomycin induced SSBs and DSBs in wt and pplig4
100
% of DNA damage remaining
80
wt
60
40
pplig4
Bleomycin treatment 1 hour
wt
SSBs
A/N
pplig4 A/N
2 μg/ml
DSBs 30 μg/ml
20
0
0
60 120 180 240 300 360
t (minutes)
Holá et al. 2013
Repair of DSBs in wt and pprad50
Bleomycin 30 μg/ml
treatment 1 hour
Kamisugi et al. 2012
Fast and slow DSB repair
Goodarzi et al 2010, Goodarzi & Jeggo 2011
Repair kinetics of DSBs in Arabidopsis, Physcomitrella and
human HEK cells
Selection of APT- mutantants
Inactivsted Adenine phosphoribosyltransferase (APT) provides resistance to 2FA
(2-fluoroadenine and as such enables positive selection of mutants
-
One day old protonemata at the
stage of 4 to 7 cells are exposed
to mutagens
-
Treated tissue is plated on plates
with 2FA
-
Selection of 2FA resistant foci
-
Isolation of genomic DNA and
PCR amplification of APT locus
-
Sequencing of entire APT locus
with 5’ and 3’ untranslated control
regions
Mutations identified in APT-
Conclusions:
1. Comet assay is applicable to various plant species and tissues. CA is quick
and enables direct determination of rapid repair kinetic, difficult to access by
other technique. She is a tool !
1. Number of viable repair mutants exists in model plants Arabidopsis thaliana
and moss Physcomitrella patens, which enable to study repair responses
inaccessible e.g. in mammalian systems. Dividing, growing and fully
differentiated tissues are available for comet assay analysis.
2. Comet assay does not have a memory and just reflects current state. Other,
auxiliary methods are often needed to support or explain Comet data.
In colaboration with:
CPS, Faculty of Biological Sciences, University of Leeds, UK
Christopher E. West, Wanda M Waterworth, Andrew Cuming
Unite Génétique CNRS et Clermont Université, France
Charles White
ISA, Universidade Técnica de Lisboa, Portugal
José A. da Costa-Nunes
Botanik II, Universitat Karlsruhe (TH), Germany
Holger Puchta
Department of Plant Biology, University of Geneva, Switzerland
Jerzy Paszkowski
Team in our Lab – Petra Rožnovská, Pascale Schlaich
Thank you for your attention