CK2-defective Arabidopsis plants exhibit enhanced double-strand break repair rates and reduced...

CK2-defective Arabidopsis plants exhibit enhanceddouble-strand break repair rates and reduced survivalafter exposure to ionizing radiation

Jordi Moreno-Romero1,†, Laia Armengot1, M. Mar Marques-Bueno1, Anne Britt2 and M. Carmen Martınez1,*

1Departament de Bioquımica i Biologia Molecular, Universitat Autonoma de Barcelona, 08193 Bellaterra, Barcelona, Spain, and2Department of Plant Biology, University of California, Davis, CA 95616, USA

Received 21 April 2011; revised 28 March 2012; accepted 2 April 2012; published online 11 June 2012.

*For correspondence (e-mail [email protected]).†Present address: Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Center for Plant

Biology, PO Box 7080, SE-75007 Uppsala, Sweden.

SUMMARY

The multifunctional protein kinase CK2 is involved in several aspects of the DNA damage response (DDR) in

mammals. To gain insight into the role of CK2 in plant genome maintenance, we studied the response to

genotoxic agents of an Arabidopsis CK2 dominant-negative mutant (CK2mut plants). CK2mut plants were

hypersensitive to a wide range of genotoxins that produce a variety of DNA lesions. However, they were able to

activate the DDR after exposure to c irradiation, as shown by accumulation of phosphorylated histone H2AX

and up-regulation of sets of radio-modulated genes. Moreover, functional assays showed that mutant plants

quickly repair the DNA damage produced by genotoxins, and that they exhibit preferential use of non-

conservative mechanisms, which may explain plant lethality. The chromatin of CK2mut plants was more

sensitive to digestion with micrococcal nuclease, suggesting compaction changes that agreed with the

transcriptional changes detected for a number of genes involved in chromatin structure. Furthermore, CK2mut

plants were prone to transcriptional gene silencing release upon genotoxic stress. Our results suggest that CK2

is required in the maintenance and control of genomic stability and chromatin structure in plants, and that this

process affects several functions, including the DNA damage response and DNA repair.

Keywords: protein kinase CK2, Arabidopsis thaliana, DNA damage responses, comet assay, MNAse sensitivity.

INTRODUCTION

The integrity of DNA is continuously challenged within the

cell. To counteract the severe biological consequences of

DNA damage, an intricate network of genome surveillance

mechanisms, often referred to as DNA damage responses

(DDRs), has evolved. Most of the DDR components identified

in animals and yeasts have counterparts in plants (Britt,

1996, 1999; Bray and West, 2005; Kimura and Sakaguchi,

2006). However, DNA repair pathways in animals include

some components for which homologues have not been

found in plants, and some DDR regulators are unique to

plants (Britt, 1999; Kimura and Sakaguchi, 2006; Yoshiyama

et al., 2009).

The initial stages of the DDR in mammals are governed by

a pair of closely related protein kinases, termed ataxia

telangiectasia mutated (ATM) and ATM- and RAD3-related

(ATR). ATM and ATR function as sensors of DNA damage,

and control the phosphorylation of histone H2AX in regions

close to damaged chromatin (Kinner et al., 2008; Mah et al.,

2010). Phosphorylated H2AX (c-H2AX) mediates the forma-

tion of DNA damage foci, which are large aggregates of

proteins and repair factors that surround the lesion sites

(Riches et al., 2008). Plants possess ATM and ATR ortho-

logues (Garcia et al., 2003; Culligan et al., 2004). ATM senses

double-strand breaks (DSBs), triggering a transcriptional

response to ionizing radiation (IR) (Garcia et al., 2003),

whereas ATR senses repair intermediates or stalled replica-

tion forks and has a prominent role in the UV-induced

response (Culligan et al., 2004; Yoshiyama et al., 2009;

Furukawa et al., 2010). Both ATR and ATM are involved in

IR-induced phosphorylation of H2AX in Arabidopsis,

although ATM is responsible for the majority of focus

formation in M-phase cells (Friesner et al., 2005). IR-induced

DSBs are among the most harmful lesions in DNA, and must

therefore be eliminated before chromosome segregation. In

ª 2012 The Authors 627The Plant Journal ª 2012 Blackwell Publishing Ltd

The Plant Journal (2012) 71, 627–638 doi: 10.1111/j.1365-313X.2012.05019.x

eukaryotes, DSBs can be repaired by two major pathways:

homologous recombination (HR) and non-homologous

DNA end joining (NHEJ). HR is a high-fidelity mechanism

that uses unbroken, homologous sequences to template

repair of DSBs. It is mostly used to repair DSB breaks due to

DNA replication, using sister chromatid information (Orel

et al., 2003). In contrast, NHEJ does not require significant

sequence homology for DSB repair, and is a prominent

mechanism in eukaryotes (Lieber, 2010). In plants, four

major non-homologous recombination pathways have been

described: (i) canonical non-homologous end joining

(C-NHEJ), which is Ku-dependent, (ii) alternative end joining

(A-EJ or A-NHEJ), which is more error-prone and uses

microhomologies for recombination (also called MMEJ), (iii)

the back-up pathway (B-NHEJ), which is Ku-independent

and involves Parp1, Xrcc1 and DNA ligase III, and (iv) an

unidentified pathway that confers severe genomic instability

(Charbonnel et al., 2011). A hierarchical organization of

these pathways during post-S phase has been proposed in

Arabidopsis (Charbonnel et al., 2011).

DNA damage responses are tightly linked to chromatin

structure. In plants, mutations in replication-coupled chro-

matin assembly factor or chromatin assembly factor 1 (CAF-

1) result in sensitivity to DNA damage, de-repression of

transcriptional silencing, and genome instability (Takeda

et al., 2004; Kirik et al., 2006; Schonrock et al., 2006). Chro-

matin is also a major factor in the regulation of the HR

pathway, as exemplified by different Arabidopsis mutants,

such as fas1-4, which is defective in the p150 subunit of CAF-

1, mim, which lacks a chromatin structural component

related to the structural maintenance of chromosomes

(SMC) family (Mengiste et al., 1999; Kirik et al., 2006), and

bru1, which encodes a nuclear protein of unknown function

(Takeda et al., 2004).

Protein kinase CK2 is an evolutionarily conserved Ser/Thr

kinase involved in a wide variety of cellular functions

(Issinger, 1993; Allende and Allende, 1995; Meggio and

Pinna, 2003), including the DDR (Loizou et al., 2004). Inter-

estingly, most DDR-related CK2 substrates in mammals are

proteins that facilitate access to DNA or form a scaffold to

support complexes involved in chromatin remodelling.

Examples include HP1b, a heterochromatin-binding protein

that facilitates access of DDR factors (Ayoub et al., 2008,

2009), XRCC1, a scaffold protein that recruits the machinery

for single-strand break (SSB) repair (Loizou et al., 2004;

Parsons et al., 2010) and is also involved in DSB repair in

Arabidopsis (Charbonnel et al., 2010), and MDC1, an adaptor

protein that interacts with the MRN (MRE11–RAD50–NBS1)

complex involved in DSB repair (Chapman and Jackson,

2008; Melander et al., 2008; Spycher et al., 2008).

Here we have used an inducible dominant-negative allele

of CK2 to investigate the role of this protein kinase in

damage tolerance and DNA repair in the higher plant

Arabidopsis. We demonstrate that lack of CK2 activity

confers hypersensitivity to a variety of genotoxic agents,

although CK2-defective plants displayed normal levels of IR-

induced histone H2AX phosphorylation and are able to

activate a transcriptional response. Our results suggest that

mutant plants show preferential use of non-conservative

pathways for DNA repair. Moreover, changes in chromatin

structure and de-repression of transcriptional gene silencing

suggest that CK2 is required in the maintenance of chroma-

tin structure, sustaining the viability of plant cells despite the

deleterious effects of genotoxic agents.

RESULTS

Arabidopsis plants depleted of CK2 activity are hypersensi-

tive to genotoxins

In previous studies, we demonstrated that an inducible

dominant-negative allele of CK2 (CK2mut) could be suc-

cessfully used to deplete CK2 activity in Arabidopsis plants

(Moreno-Romero et al., 2008; Marques-Bueno et al., 2011).

Here we have used the same mutant to study the involve-

ment of CK2 in plant DDRs. We performed transient induc-

tions of the transgene (typically treatment of 5-day-old

seedlings with dexamethasone for 48 h), followed by

genotoxic treatment on the 7th day. Unless otherwise indi-

cated, uninduced CK2mut plants were used as controls. Our

results show that mutant seedlings are hypersensitive to IR

at 100 Gy (Figure 1a), exhibiting anthocyanin accumulation,

cotyledon necrosis and developmental arrest. The effects

are specifically due to accumulation of CK2mut protein, as

dexamethasone treatment had no effects on c-irradiated

wild-type or atm plants. The Arabidopsis atm mutant is a

well-known IR-hypersensitive mutant that is defective in the

DDR response. Moreover, CK2mut plants were hypersensi-

tive to IR when dexamethasone treatment was performed

before or after c irradiation (Figure 1b). As Arabidopsis

mutants affected in DNA repair processes show defects in

root growth under genotoxic stress (Garcia et al., 2003;

Culligan et al., 2006), we measured daily root growth in post-

IR CK2mut seedlings. Root growth was permanently

arrested upon 80 Gy treatment in CK2mut seedlings

(Figure 1c, left; note that CK2mut roots were initially shorter,

as previously described by Moreno-Romero et al., 2008),

whereas the arrest was transient in Arabidopsis wild-type.

Moreover, root viability appeared highly compromised in

CK2mut plants (Figure 1c, right).

We have recently shown that depletion of CK2 activity

alters polar auxin transport (Marques-Bueno et al., 2011). In

order to ascertain whether the IR hypersensitivity was an

indirect effect of auxin transport impairment, we treated

CK2mut and control seedlings with either indole-3-acetic

acid (IAA) or N-1-naphtylphtalamic acid (NPA) (an inhibitor

of polar auxin transport) prior to IR exposure. Quantification

of plant fresh weight in post-IR 20-day-old plants showed

that the IR hypersensitivity of CK2mut seedlings was not

628 Jordi Moreno-Romero et al.

ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 71, 627–638

reversed by exogenous IAA (Figure S1), in contrast to other

CK2mut phenotypes previously reported by Marques-Bueno

et al. (2011). Moreover, NPA slightly affected the growth of

post-irradiated control plants, but did not have deleterious

effects. Altogether, these data led us to conclude that the

hypersensitivity phenotype shown by CK2mut plants is not

an indirect consequence of impaired auxin transport.

We also tested the sensitivity of CK2mut plantlets to UV-C

and methyl methanesulfonate (MMS). CK2mut plants were

hypersensitive to UV-C radiation, showing significant

growth inhibition at 30 000 J m)2 (Figure 1d), and to MMS

over a range of 25–100 ppm (Figure 1e).

Taken together, these results strongly suggest that CK2 is

required to successfully recover from different genotoxic

treatments in Arabidopsis. The genotoxins tested are known

to produce different kinds of lesions into DNA, and thus CK2

may act upstream of different signalling pathways necessary

for DNA repair. Alternatively, CK2 may be required at multiple

points or may play a more general role in plant survival.

For the subsequent studies, we focused on IR-induced

responses using c irradiation. IR-induced responses have

been well characterized in Arabidopsis wild-type, facilitating

their study in mutant genotypes. Moreover, c irradiation has

a DSB production ratio (number of DSBs/base pair versus

dose) that is similar in all eukaryotes (Su, 2006).

CK2 expression and activity do not significantly change

after exposure to IR

The CK2a and b subunits are encoded by two small multi-

gene families. The Arabidopsis nuclear genome contains

four genes for the catalytic subunit (a) and four genes for the

regulatory subunit (b) (Salinas et al., 2006). The IR-induced

expression changes of CK2-encoding genes were very small,

never reaching twofold, whereas TSO2 (encoding the small

subunit of ribonucleotide reductase, which was used as a

positive control) was highly over-expressed under the same

conditions (Figure S2a). We also analyzed CK2 expression

data from the AtGenExpress project, which compiles Ara-

bidopsis data from ATH1 GeneChip arrays (Kilian et al.,

2007). CK2a and b transcript levels showed no significant

changes in Arabidopsis plants (18-day-old plantlets, Col-0

ecotype) treated with 1.5 lg ml)1 bleomycin (a radiomi-

metic) plus 22 lg ml)1 mitomycin C (a cross-linking agent)

(b)

(c)

atmwt CK2mut–D

ex

+D

ex

0 200 Gy50 100

–D

ex

+γ

+D

ex

+γ

+γ

+D

ex

0

1

2

3

4

0 1 2 3 4 5 6 7 8 9

Days post IR

Daily

rootgro

wth

(mm

/day)

+Dex

–Dex

(a)–D

ex

+D

ex

(d)

–D

ex

+D

ex

(e)

0 10 000 15 000 30 000 J/m2

0 25 50 100 ppm

Figure 1. Hypersensitivity of CK2mut plants to genotoxins.

(a–c) Responses to ionizing radiation (IR). Five-day-old seedlings were treated

with dexamethasone (Dex) for 48 h and then c-irradiated.

(a) Phenotype of 21-day-old seedlings after receiving 100 Gy.

(b) Dose–response experiment. IR was applied either after dexamethasone

treatment (+Dex +c) or before dexamethasone treatment (+c +Dex).

(c) Daily root growth (left panel), and cell death detected by Evan’s blue

staining (right panel) after 80 Gy IR. Scale bar = 0.3 mm.

(d) Dose–response to UV-C. Experiments were performed as in (a).

(e) Dose–response to methanesulfonate (MMS).

Images show 14-day-old seedlings.

CK2 in DNA damage responses in Arabidopsis 629


(Figure S2b). Although the plant ages and genotoxins used

in the array experiments were different from those em-

ployed in our work, both set of data support the idea that

CK2 is not transcriptionally regulated in response to DNA-

damaging agents. This is consistent with data showing that

mammalian CK2 activity is regulated mainly at a post-

translational level (Filhol and Cochet, 2009). However, no

significant changes of CK2 activity were found in c-irradiated

Arabidopsis wild-type plantlets (Figure S2c).

IR-induced global changes of gene expression in CK2mut

seedlings

Plants subjected to genotoxic stress become impaired in a

wide number of cellular functions. Particularly well known is

the IR-induced transcriptional burst in Arabidopsis plants,

affecting a large number of genes (Chen et al., 2003; Nagata

et al., 2005; Culligan et al., 2006; Kim et al., 2007; Ricaud

et al., 2007). We analyzed transcript profiles of c-irradiated

mutant plants (100 Gy) using Affymetrix ATH1 chips. The

study was performed at 1.5 h post-IR, as transcript radio-

modulation in Arabidopsis occurs as an early wave after IR

and lasts approximately 3 h, with only 10% of the genes still

showing changes 5 h post-IR (Ricaud et al., 2007). The

experimental design and comparative analysis performed

are summarized in Figure 2(a). Pairwise comparisons

revealed different numbers of affected genes depending on

the variable analyzed (Figure 2a, bottom). For instance,

induction of the transgene (pairwise comparison number 2,

‘CK2mut effect’) produced the widest changes, affecting

6614 sequences (P < 0.001) out of the 22 746 sequences

present in the array (after subtracting the changes obtained

in plants transformed with the empty vector, shown in

comparison number 1). IR-induced transcriptional changes

in control plants (pairwise comparison number 3, ‘gamma

effect’) correlated well with those reported in the literature

for Arabidopsis wild-type plants (Table S1a).

Genes showing IR-induced transcriptional changes in

control and mutant seedlings were grouped into GO func-

tional categories, depicted in Figure 2(b) (‘gamma effect’

and ‘CK2mut + gamma effect’, respectively). The number of

genes within each category is shown in Table S1a. There are

four shared categories, but the number of genes within them

and/or their statistical significance differ between the two

conditions. Ninety genes were present in both datasets

(‘CK2mut + gamma effect’ and ‘gamma effect’), 89 showing

positive correlation (59 up-regulated and 30 down-regulated

in both cases) and one showing negative correlation (up-

regulated in ‘CK2mut + gamma effect’ and down-regulated

in ‘gamma effect’) (Figure 2c, left, and Table S1b). The IR-

induced fold changes in expression were lower in mutant

than control plants (see the slope of the correlation plot in

Figure 2c, right), and this was confirmed by RT-PCR for

some of these genes (Figure S3). A time-course study

showed that gene under-expression in the mutant was due

to weaker induction not induction delay (Figure 2d).

To gain insight into the nature of the IR-induced tran-

scriptional response, we further analyzed the genes specif-

ically involved in DNA repair processes. One hundred and

fifty genes have a known or predicted function in DNA repair

(http://www.uea.ac.uk/~b270/repair.htm). Some are radio-

modulated (rapidly IR-induced) (Schonrock et al., 2006;

Dohmann et al., 2008), although the majority are not clearly

induced after DNA damage (http://bar.utoronto.ca/ or http://

www.weigelworld.org/resources/microarray/AtGenExpress).

DNA repair genes with significant changes in our experi-

mental conditions (ANOVA, P value <0.001) were classified

into DNA repair pathways. Many of those genes were up- or

down-regulated in non-irradiated CK2mut seedlings,

although the fold changes were rather modest (Figure 2e

and Table S1c). In particular, several of the genes involved in

HR appeared to be transcriptionally repressed, but no major

changes were found in the genes of the NHEJ pathways.

Analysis of array data from other authors (Menges et al.,

2005) showed that genes involved in HR are cell cycle-

regulated, with peaks of expression at S phase, whereas

genes involved in NHEJ pathways show steady-state expres-

sion levels during the cell cycle (Figure S4a). We have

previously demonstrated that CK2mut gene expression

leads to cell-cycle arrest at G1/S (Moreno-Romero et al.,

2008), which agrees with the expression profile of cell

Figure 2. Transcript profiling of IR-induced responses in CK2mut seedlings.

(a) Experimental design and scatter graphs. Pairwise comparisons are numbered 1 to 4; the corresponding scatter graphs show the number of up-regulated genes

(red) and down-regulated genes (green). ANOVA P value <0.001.

(b) BiNGO gene ontology (GO) enrichment analysis of IR-induced responses in dexamethasone-treated CK2mut plants (right) and untreated CK2mut plants (left).

Output categories (P value <0.05) are represented by coloured circles: red/pink, up-regulated genes; green, down-regulated genes (bi-colour circles include both

over- and under-expressed genes). Circle sizes are proportional to the number of genes. Circle perimeters represent the results of comparing the corresponding

expression data in each condition with those in dexamethasone-treated non-irradiated CK2mut plants (‘CK2mut effect’). Unmarked perimeters mean that the sub-

categories were absent in ‘CK2mut effect’, and black, white or dashed perimeters mean relative over-expression, under-expression or both, respectively. The GO

categories were subsequently grouped into higher-order functions (pale blue areas), which are indicated in bold when present in the two conditions, in italics when

present only in ‘gamma effect’, or underlined when present only in ‘CK2mut + gamma effect’.

(c) Venn diagrams and correlation plot obtained by comparison between datasets 3 and 4.

(d) Validation of transcriptional changes by semi-quantitative RT-PCR. Values were normalized to those of the EF1A (at5g60390) gene. Values are means � SD of

three biological triplicates. a.u., arbitrary units.

(e) Fold-change analysis of DNA repair genes. Genes showing significant changes (ANOVA, P value <0.001) were classified into different DNA repair pathways and

pairwise comparisons were performed [shown in (a) as 2 (+Dex), 3 (–Dex +c) and number 4 (+Dex +c)].



Administrator

Resaltado

Administrator

Resaltado

Administrator

Resaltado

MPG At3g12040

TagI - E. coliAt5g44680

At1g75090

At1g80850

APE1 At3g48420

RAD51 At5g20850

DMC1 At3g22880

RecA – E. coli At3g10140

BRCA1 At4g21070

At1g04020

RAD50 At2g31970

BLM At1g10930

RecQ – E. coli At4g35740

adventitious root development

Carboxilic acid metabolic process (JA biosynthetic

process)

NO biosynthetic process

IAA biosynthetic

process

Glycosinolate biosynthetic process

Vitamin E biosynthetic process

Regulation of

telomerase

activity

Translation

Removal of

superoxide radicals

DNA repair

Cell cycle

Response to stress

Nucleosome assembly

DNA metabolic process

Response to biotic stimulus

‘Gamma effect’ ‘CK2mut + Gamma effect’(b)

(c)

30

172

72

DOWN

‘Gamma

59

203

53

UP

‘Gamma

‘CK2mut +Gamma

‘CK2mut +Gamma

Empty vector–Dex

CK2mut–Dex

CK2mut–Dex

+γ

Empty vector+Dex

CK2mut+Dex

CK2mut+Dex

+γ

1

2

3 4

(a)

1

2

4

3 ‘Gamma effect’

‘CK2mut + Gamma effect’

‘Empty vector effect’

‘CK2mut effect’

(e)

Correlation plot

‘Gamma effect’ fold change

‘CK

2m

ut

+G

am

ma

effect’

fold

change

5

10

15

20

0 5 10 15 20

TSO2

RAD51PARP1

BRCA1

59 genes

UP

correlated

30 genesDOWN

correlated

(d)

TSO2

1

2

3

RAD51

1

2

3

4

PARP1

4

8

12

16

−Dex +Dex

0 15 3 6 12 0 15 3 6 12

Expre

ssio

nle

vel(a

.u.)

2

4

6

8CYCB1;1

2

4

6

8BRCA1

–1.5 0 1.5

Log10 ratio

Mismatch repair

Homologous recombination

MSH6 At4g02070

RPA1

At4g19130

At5g45400

At5g08020

At5g61000

RPA2 At2g24490

GTF2H1 At1g55610

ERCC1 At3g05210

POLE1 At1g08260

PCNA At2g29570

At1g07370

Mfd - E. coli At3g02060

Phr – E. coli At1g04400

At2g47590

Nucleotide excission repair

+D

ex

–D

ex

+γ

+D

ex

+γ

Base excissionrepair

Photorepair

nucl. pool

rad6 pathway

at2g31320

at4g02390PARP

Poly(ADP-ribose) polymerase (PARP) enzymes

At3g46940DUT

RAD6A

MMS2

UBC13

At1g23260

At1g78870

At2g02760Time after γ(h)

467

144

3420

3611

262

202

112

102

3106

3508

. .

+D

ex

–D

ex

+

+D

ex

+

γ γ

effect’

effect’

effect’effect’



cycle-regulated genes in mutant seedlings (array data,

Figure S4b). Therefore, transcriptional under-expression of

the HR pathway in mutant plants may be due to G1/S cell-

cycle arrest. In spite of this, our array data also show that

radio-modulated genes involved in the HR pathway (such as

RAD51 or BRCA1) were IR-induced in mutant plants (Fig-

ure 2e), indicating that the pathway may be activated in a

cell cycle-independent manner under stress conditions.

CK2mut seedlings show high DNA repair proficiency

The DNA repair proficiency of mutant seedlings was inves-

tigated by comet assays. The amount of DNA in the tail

(Figure 3a) was measured before and after treatments with

two different genotoxins (bleomycin or IR), using two dif-

ferent protocols (the N/N protocol that detects DSBs, and the

A/A protocol that detects DSBs, SSBs and the majority of

labile sites; Menke et al., 2001). Surprisingly, comet assays

performed on bleomycin-treated plants with the N/N proto-

col showed that DSBs were more rapidly repaired in mutant

than in control plants (Figures 3b and S5a). Similar results

were obtained using the A/A protocol in combination with cirradiation (100 Gy): the tail of the comets disappeared faster

in mutant plants (Figure 3c). As a negative control, we used

the IR-hypersensitive atm mutant, which did not show en-

hanced DNA repair rates in our assays (Figure S5a,b). These

results suggest that mutant plants are more proficient in

repairing DSBs and other secondary lesions produced by IR

or bleomycin, despite their hypersensitivity to these agents.

CK2-defective plants show decreased homologous

recombination

To analyze the efficiency of DSB repair by homologous

recombination (HR), we used two recombinogenic substrates

present in Arabidopsis transgenic lines generated by H. Puc-

hta (Botanisches Institut, Universitat Karlsruhe, Germany).

HR events were detected by restoration of b-glucuronidase

(GUS)activity,whichwasmeasuredasablueprecipitate (Orel

et al., 2003). CK2 activity was inhibited prior the assay by

(d)

GUS

GU

GU US

US20

40

− γ

HR

events

/leaf

+γ

−TBB+TBB

10

20

− γ +γ

HR

events

/leaf

GUUS

GUUSU

+ U

GUSUU

U

DGU.US line

DU.GUS line

Damage

Repair

(a)

(b) (c)

0

20

40

60

80

100

0 20 600

20

40

60

80

100

0 0.5 1.5 4

*

** *

+Dex

(e)

(f)

5

10

15

20

–γ +γ

Mock+TBB

+aph+aph +TBB

HR

events

/leaf

*

* **

0

2

4

6

8

Fre

sh

we

ight

per

pla

nt(m

g)

*

*

*

Mock+Dex+aph

–Dex

+Dex

–Dex

Time after bleomycin treatment (min) Time after gamma treatment (h)

%D

NA

inta

il

%D

NA

inta

il

*

*

*

*Mock +Dex

+aph

Figure 3. DNA repair proficiency.

(a) Comet functional assay.

(b) Comet assays in neutral conditions (N/N

protocol). Top panel: images from a typical

experiment. Bottom panel: bar plots showing

the percentage tail DNA in the population of

nuclei. Seedlings were incubated with 50 lM

bleomycin for the indicated times. Values are

means � SD of at least three experiments.

(c) Comet assays in alkali solution (A/A protocol).

Seedlings were irradiated with 100 Gy. Data are

represented as in (b).

(d) Effect of CK2 inhibition on intra-chromosomal

HR frequency. Left: the two types of constructs

used. Right: number of blue spots (HR events/

leaf) in non-irradiated (–c) and 24 h post-irradi-

ated seedlings (+c) (n ‡ 6).

(e) Effect of cell-cycle arrest on intra-chromo-

somal HR frequency. DU.GUS plantlets were

incubated with 10 lg ml)1 aphidicolin for 24 h

and then c-irradiated. GUS staining was carried

out 3 days after c irradiation.

(f) Effect of cell-cycle arrest on plants’ sensitivity

to IR. Five-day-old CK2mut plants were treated

with either ethanol (mock), dexamethasone

(Dex) or aphidicolin (aph) for 48 h and then

c-irradiated (100 Gy). Images and fresh weights

are from 20-day-old plantlets submitted to the

indicated treatments.

Asterisks indicate significant differences. Statis-

tical analysis was performed using Student’s

t test at P < 0.005 (b–e) or the non-parametric

Kruskal–Wallis test at P < 0.05 (f).



incubation of plants with tetrabromobenzotriazol (TBB)

(Shugar, 1994), a specific inhibitor that does not interfere with

the activity of other Arabidopsis kinases (Espunya et al.,

1999). TBB-treated seedlings exhibited fewer blue sectors

than control plants in all the conditions tested (Figure 3d),

indicating lower HR frequency. However, the number of blue

sectors significantly increased upon IR (blue spots were seen

24 h after IR), both in control and TBB-treated seedlings,

suggesting that CK2 is not required to activate the HR path-

way. In order to check whether the lower HR efficiency of TBB-

treated plants was due to an indirect effect of cell-cycle arrest

produced by CK2 inhibition, we assessed the effect of aphi-

dicolin, a drug that blocks the cell cycle at G1/S, on HR profi-

ciency. Figure 3(e) shows that, in non-irradiated seedlings,

the number of HR events was similarly decreased in the

presence of either aphidicolin or TBB (blue spots were seen

72 h after IR). Moreover, c irradiation induced significant

higherHR activation inaphidicolin-treatedplants than inTBB-

treated plants (1.85-fold versus 1.47-fold, respectively), and

the simultaneous presence of TBB and aphidicolin did not

have an additive effect on HR activation. To assess the long-

term effects of aphidicolin on plants’ sensitivity to IR, plants

were irradiated with 100 Gy and fresh weights were mea-

sured13 daysafter IR.Figure 3(f) showsthat IRaffectsgrowth

significantly more in CK2-defective plants than in aphidicolin-

treated plants. Moreover, aphidicolin-treated plants were

able to develop true leaves, whereas CK2-defective plants

were developmentally arrested. As previously demonstrated,

a 100 Gy dose is lethal for CK2mut plants, but not for aphi-

dicolin-treated plants. We conclude that, although cell-cycle

arrest may contribute to the weaker HR activity in non-irradi-

ated CK2-defectiveseedlings, additional mechanismsmay be

responsible for the impaired DDR in the same plants.

IR-induced histone H2AX phosphorylation and cyclin

CYCB1;1 expression are unaffected in CK2mut plants

One of the first molecular events induced by the DDR is

phosphorylation of histone H2AX. To determine whether

this mechanism was activated in CK2mut plants, protein

extracts from c-irradiated (100 Gy) seedlings were prepared

at 20 min after IR, and immunoblotted with an antiserum

specific to the phosphorylated form of H2AX (c-H2AX). We

detected a band of the expected molecular weight, the

intensity of which was similar in mutant and control plants

(Figure 4a). This finding was corroborated by performing a

time-course analysis of c-H2AX accumulation: both mutant

and control plants showed the maximum band intensity at

20 min after IR, rapidly decreasing afterwards (Figure 4b).

Another molecular marker of DDR activation is up-regu-

lation of mitotic cyclin CYCB1;1 (Culligan et al., 2006).

CYCB1;1 accumulation was monitored during 3 days post-

IR (100 Gy), using the translational fusion CYCB1;1::GFP

introduced into the CK2mut background. GFP fluorescence

was similar in c-irradiated mutant and control plants

(c)

4

8

12

0 4 24 48 72 0 4 24 48 72

–Dex +Dex

Root

tip

fluore

scence

(a.u

.)

0.2

0.6

1.0

0.3 hours post-IR

Band

inte

nsity

(a)

wt

132

78

45.7

18.4

32.5

7.6

H2A

X-P

0 0.3 0 0.3

– Dex + Dex

0

0.5

1.0

1.5

2.0

2.5

0 0.3 4 12

wt

(b)0 0.3 4 12 0 0.3 4 12

18.4

18.4

– Dex +Dex

CK2mut

wt

h post-IR

h post -IR

ratio

–D

ex/+

Dex

Band

inte

nsity

ratio

–D

ex/+

Dex

h post -IR

Figure 4. c-H2AX accumulation and CYCB1;1 expression in response to IR.

(a,b) Immunoblot detection of c-H2AX. Plants received 100 Gy of IR and then

were returned to growth chambers and harvested 20 min after removal from

the gamma source (a) or at the indicated times (b). Images shown are from a

typical experiment of three. A Coomassie-blue stained gel is shown in (a) (left

panel). The bottom panels in (a) and (b) show quantification of c-H2AX band

intensities, represented as )Dex/+Dex ratios, using data from three different

experiments (means � SD). The horizontal lines show the corresponding

ratios for wild-type plants.

(c) Time course of CYCB1;1-GFP expression in root tips. GFP fluorescence was

monitored and quantified (bar plots) at the indicated times. a.u., arbitrary

units.



(Figure 4c), although the initial CYCB1;1 levels were lower in

the mutant, as reported previously (Moreno-Romero et al.,

2008).

These data indicate no significant changes in the initial

responses of CK2mut plants to IR-induced DNA damage,

confirming that their phenotype of hypersensitivity is not

due to defects in the activation of signalling pathways.

Chromatin structure in CK2mut plants

Due to the close relationship between chromatin structure

and the DNA damage response, we assessed the sensi-

tivity of the chromatin of CK2mut plants to micrococcal

nuclease (MNase). MNase is able to produce nicks in the

inter-nucleosomal DNA, and its efficiency depends on the

degree of chromatin compaction. Figure 5(a) shows that

the chromatin from CK2mut plants was more sensitive to

MNase digestion than the chromatin from control plants

(Figure 5a). This was confirmed by performing a time-

course analysis with 2.5 U ml)1 of nuclease at 4�C (Fig-

ure 5b) or 37�C (Figure S6a). In all the conditions tested,

nuclei from CK2mut plants were digested faster, leading to

accumulation of DNA bands of lower molecular weight

(Figure S6b). Several laboratories have demonstrated a

genetic link between chromatin structure, DNA repair and

transcriptional gene silencing (Takeda et al., 2004; Elmayan

et al., 2005). The array data from CK2mut seedlings

showed significant transcriptional changes in genes in-

volved in chromatin structure, such as histone modifica-

tion, heterochromatin formation or nucleosome assembly

(Figure S7a). Moreover, determination of transcript levels

of FAS1 and FAS2 (encoding two subunits of the CAF-1

complex) and of MIM by RT-PCR showed that they were

under-expressed in mutant seedlings, whereas those of

MSI1 (the third subunit of CAF-1) did not show significant

changes (Figure 5c). It has been reported that loss-of-

function fas1, fas2 and mim mutants are hypersensitive to

MMS, and that fas1 and fas2 mutations activate the

transcriptionally silent information (TSI) element (Mengiste

et al., 1999; Takeda et al., 2004). TSI repeats, concentrated

in pericentromeric chromosomal regions, are heavily

methylated and silenced in wild-type Arabidopsis, and

their expression is activated in some mutants affected in

epigenetic regulation (Steimer et al., 2000). In contrast,

CK2-defective plants show normal basal TSI transcript

levels. Upon IR, TSI levels increased 13.5-fold in CK2mut

plants and only 2.8-fold in control plants (Figure 5d). In

order to assess whether the IR-induced TSI over-expres-

sion in CK2mut plants was an indirect effect of the altered

auxin responses reported for this mutant (Marques-Bueno

et al., 2011), we measured TSI levels in post-IR plants

previously incubated with either the synthetic auxin ana-

logue 1-naphthaleneacetic acid (NAA) or the auxin trans-

port inhibitor NPA. Figure 5(e) shows that neither

compound affects TSI levels in wild-type plants, indicating

that impairment of auxin transport is likely not responsible

for the TSI release of silencing. Interestingly, incubation

with NPA increases TSI over-expression and IR sensitivity

in CK2mut plants (Figures 5e and S1, respectively).

(a) MNase (U mL–1)–Dex +Dex

0 1 5 10 20 40 0 1 5 10 20 40g

gc/g0 gt/g01.00

Conc.

0.89

0.21

0.12

0.04

0.03

1.00

0.70

0.08

0.01

0.00

0.00

(b) MNase (hours)

0 0.5 2 2.5 0 0.5 2 2.5–Dex +Dex

gTime

1.00

1.06

1.08

0.68

1.00

0.72

0.25

0.15

(d)(c)

0

2

4

6

8

Exp

ress

ion

leve

l (a.

u.)

FAS1MIM FAS2 MSI10

2

4

0 1 7Days post-IR

(e)

0

2

4

IAANPA

––

+–

–+

––

+–

–+

Exp

ress

ion

leve

l (a.

u.)

Exp

ress

ion

leve

l (a.

u.)

+Dex–Dex

+Dex–Dex

+Dex–Dex

Figure 5. Chromatin structure.

(a,b) Chromatin digestions with micrococcal nuclease (MNase). Gel electrophoresis of digested nuclei with different concentrations of MNase for 15 min at 37�C (a)

or with 2.5 U ml)1 of MNase at 4�C for the indicated times (b). The intensities of genomic bands at time 0 (g0), or after digestion with variable concentrations of

MNase (gc) or for different incubation times (gt) were determined by gel scanning.

(c) Expression levels of sequences related to chromatin-structure maintenance, measured by semi-quantitative RT-PCR.

(d,e) TSI expression. TSI transcript levels were measured by quantitative RT-PCR at the indicated times after IR (100 Gy IR) (d), or at 13 days after IR in plants

previously incubated with either 1 lM IAA or 10 lM NPA (e). Expression data were normalized to those of EF1A. Values are means � SD of three biological

replicates. a.u., arbitrary units.



Administrator

Resaltado

Administrator

Resaltado

Administrator

Resaltado

In addition, microarray data showed that gene silencing in

either telomeric regions (data were analyzed as in Schonrock

et al., 2006, for telomeric genes present in the array),

pericentromeric regions of chromosomes 2 and 4 (Nakahig-

ashi et al., 2005), or transposable-related elements, was

maintained in CK2mut plants (Figure S7b,c,d, respectively).

We could not analyze gene expression in heterochromatic

regions in c-irradiated CK2mut plants, because the array

data were obtained at 1.5 h post-IR, which is too short a time

to detect such changes. However, the above data enabled us

to conclude that CK2mut plants have an altered, less

compacted chromatin structure, and that these plants show

de-repression of gene silencing in the presence of stressors.

DISCUSSION

Studies in yeast and mammals suggest that the protein ki-

nase CK2 is involved in the DNA damage response. This was

supported by experimental data showing that CK2 mutants

or inhibition of CK2 activity caused hypersensitivity to DNA-

damaging agents. Moreover, key proteins of the DDR

machinery have been shown to be phosphorylated by CK2

(Krohn et al., 2003; Koch et al., 2004; Loizou et al., 2004;

Cheung et al., 2005; Yamane and Kinsella, 2005). The Ara-

bidopsis mutant used in this work (CK2mut plants) exhibits

hypersensitivity to a broad range of genotoxins, such as IR,

UV-C, bleomycin and MMS. Moreover, like the much-stud-

ied Arabidopsis atm mutant (Garcia et al., 2003; Culligan

et al., 2006), CK2mut root tips do not recover from exposure

to 100 Gy IR, and, instead, terminally differentiate into root

hair-producing organs. Interestingly, hypersensitivity to IR

was detected when CK2 activity was depleted either before

or after irradiation, suggesting that CK2 participates in long-

term DNA damage responses and/or survival.

Unexpectedly, comet assays showed that CK2mut plants

exhibited enhanced proficiency in repairing IR- and bleomy-

cin-induced DSB lesions. DSB repair involves two different

pathways: homologous recombination (HR) and non-homol-

ogous end joining (NHEJ). The efficiency of the HR pathway

was checked by using two recombinant substrates, DGU.US

and DU.GUS, which exploit restoration of GUS marker gene

by two alternative mechanisms: Rad51-independent (for

DGU.US) and Rad51-dependent (for DU.GUS). CK2-defective

plants showed a lower number of HR events with either

substrate in both irradiated and non-irradiated plants. This

supports the idea that HR is less active in CK2-defective

plants, and that the rapid DSB repair activity measured by

comet assays is due to enhanced NHEJ mechanisms. Our

experiments with aphidicolin demonstrated that cell-cycle

blockage at G1/S has a clear negative impact on HR activa-

tion, but this is not sufficient to explain the lower number of

HR events in post-irradiated CK2-defective seedlings.

According to the hierarchical action of DSB repair pathways

proposed by Charbonnel et al. (2011), HR is a late-onset

pathway that repairs DSBs that were not processed by NHEJ.

As CK2mut plants show very rapid DNA repair kinetics, a

possible explanation is that most breaks are repaired by

NHEJ pathways, which involves the use of non-conservative

mechanisms and can lead to loss of genetic material and/or

accumulation of errors that may be deleterious. In particular,

the still unidentified fourth pathway postulated by Charbon-

nel et al. (2011) that confers severe genomic instability might

be favoured in CK2mut plants.

ATM-dependent phosphorylation of histone H2AX, one of

the earlier responses to DSB induction, is conserved in

plants (Friesner et al., 2005). The kinetic of H2AX phosphor-

ylation/dephosphorylation in IR-treated CK2mut plants

strongly suggests that both sensing of the stimulus and

activation of the early steps in the signalling cascade are not

affected by loss of CK2 activity. This is concordant with the

observation that radio-modulated genes are induced at the

appropriate times in the mutant, although to a lesser extent

than in wild-type plants. According to some authors, inhi-

bition of CK2 activity in mammals decreases the levels of c-

H2AX and the number of repair foci (Ayoub et al., 2008).

However, other authors have recently reported that CK2

inhibition only delays c-H2AX foci removal, reducing sur-

vival of irradiated mammal cells (Zwicker et al., 2011). In

either case, CK2 in mammals appears to be involved in

modulating c-H2AX levels, whereas our results suggest that

this is not the case in Arabidopsis. In mammals, CK2-driven

phosphorylation of MDC1 is essential for accumulation and

retention of the MRN complex and contributes to the

propagation of c-H2AX (van Attikum and Gasser, 2009).

Although homologous genes for the MRN complex have

been cloned in Arabidopsis and shown to play an important

role in DNA repair and meiotic recombination (Waterworth

et al., 2007), no plant homologues for MDC1 have been

identified so far in plants. Therefore, animals and plants may

differ mechanistically with regard to this process.

Chromatin dynamics is linked to DNA repair: minutes after

damage, important changes in chromatin compaction occur,

which are facilitated by histone modifications and chromatin

remodelling (van Attikum and Gasser, 2005; Groth et al.,

2007). In plants, many of the Arabidopsis mutants exhibiting

hypersensitivity to genotoxic treatments are also affected in

chromatin structure, such as fas1 and fas2 (Kirik et al., 2006),

bru1 (Takeda et al., 2004), mre11 (Bundock and Hooykaas,

2002), mim (Mengiste et al., 1999), and npr1 and npr2 (Zhu

et al., 2006). Some of these mutants also show de-repres-

sion of heterochromatic genes (Elmayan et al., 2005). The

chromatin of CK2-defective plants was hypersensitive to

MNase digestion, indicating either less compaction or

structural disorganization. Moreover, the expression of

genes such as FAS1 or MIM (which are involved in chroma-

tin remodelling) was decreased, supporting the idea that

CK2 activity regulates components directly involved in

chromatin structure. However, transcriptional gene silenc-

ing was maintained in the heterochromatic structures ana-



lyzed (TSI, transposons and pericentromeric/telomeric re-

gions), as late as 2 days after dexamethasone treatment,

but, interestingly, TSI transcript levels increased dramati-

cally when CK2mut seedlings were subjected to genotoxic

stress. In this sense, CK2mut plants behaved similarly to

npr1 and npr2 mutants, which show normal basal TSI levels

but over-expression of this locus after genotoxic treatments.

NPR1 and NPR2 encode histone chaperones that act in the

maintenance of dynamic chromatin (Zhu et al., 2006).

In conclusion, our results show that CK2-defective Arabid-

opsis plants are hypersensitive to a wide range of genotoxic

stress, suggesting that, as occurs in mammals, CK2 modu-

lates the DDR in plants. CK2-defective plants are able to

activate the DDR machinery, but preferentially use non-

conservative mechanisms for DNA repair, which may explain

plant lethality. The enhanced activity of NHEJ pathways may

be facilitated in these plants because, in contrast to the HR

pathway, expression of genes involved in NHEJ pathways is

not cell cycle-dependent, and thus is unaffected by depletion

of CK2 activity. Moreover, DNA damage sites may be more

accessible to repair factors in CK2mut plants due to their less

compacted chromatin, contributing to and amplifying the

rapid use of NHEJ pathways instead of HR. In this scenario,

CK2, by virtue of its dual activity as a regulator of chromatin

remodelling and cell-cycle progression, plays a role in

protecting cells from IR by avoiding extensive use of error-

prone DNA repair mechanisms that, although quickly repair-

ing the DNA, have deleterious effects.

EXPERIMENTAL PROCEDURES

Plant material

Growth of plants, in vitro germination and culture, and generation ofCK2mut and CYCB1;1::GFP x CK2mut transgenic plants have beendescribed by Moreno-Romero et al. (2008). Seeds containing therecombinogenic substrates DGU.US and DU.GUS were kindly pro-vided by Dr H. Putcha (Botanisches Institut, Universitat Karlsruhe,Germany). Expression of the CK2mut transgene was induced with1 lM dexamethasone dissolved in ethanol. Root lengths weremeasured on scanned plant images using a GS-700 imaging den-sitometer (Bio-Rad, Hercules, CA, USA) and IMAGEJ software (http://rsb.info.nih.gov/ij). Viability of root tip cells was tested by incubationwith Evans blue (0.25% w/v) (Sigma-Aldrich, St Louis, MO, USA) for1 h as described by Gaff and Okong’o-Ogola (1971). CYCB1;1::GFPexpression was monitored with a DMRB microscope (Leica Micro-systems GmbH, Wetzlar, Germany) equipped with a Leica DC200digital camera. Quantification of GFP fluorescence was performedusing LEICA Q500MC software, in non-saturating conditions. IAA,NAA and NPA (Sigma-Aldrich) were dissolved in ethanol at the gi-ven concentrations and added to Petri dishes. Aphidicolin (Sigma-Aldrich) was dissolved in dimethylsulfoxide and used at 10 lg ml)1

for the indicated times.

Genotoxic treatments

Unless otherwise indicated, genotoxic treatments were performedon 7-day-old seedlings grown on MS plates or liquid medium.Ionizing radiation (IR) was provided by a 137caesium source gener-ated by an IBL 437C irradiator (CIS Bio International, Bagnols/Ceze,

France) at a dose rate of 5.7–5.5 Gy min)1. UV-C radiation wasprovided by a Stratalinker UV crosslinker (Stratagene, AgilentTechnologies, Inc., Santa Clara, CA, USA). Bleomycin treatmentswere carried out for 1 h on 7-day-old seedlings grown on plates.MMS treatments were carried out for 7 days on 6-day-old seedlingsgrown in MS liquid medium and previously incubated with dexa-methasone or ethanol for 24 h.

RT-PCR assays

RNA extraction and quantitative RT-PCR assays were performed asdescribed by Moreno-Romero et al. (2008). For semi-quantitativeRT-PCR, the linear range of PCR product synthesis was establishedfor each primer pair, and the number of cycles was chosenaccordingly. Primer sequences and annealing temperature areshown in Table S2.

Hybridization and analysis of ATH1 Affymetrix arrays

Total RNA was isolated from frozen tissue samples using Trizol(Invitrogen, Carlsbad, CA, USA), and later purified using QiagenRNeasy Plant Mini Kit columns (Qiagen, Valencia, CA, USA). Threeindependent RNA preparations were made from pooled samples foreach of the conditions and lines. Microarray hybridizations (Gene-Chip� ATH1 from Affymetrix (Affymetrix, Inc., Santa Clara, CA,USA), with 22 810 sequences) were performed by the genomicfacilities of Progenika Biopharma (Derio, Spain) using the method-ology and equipment recommended by Affymetrix Inc. The com-plete array data for plants transformed with the empty vector (withand without dexamethasone) have been submitted to the Notting-ham Arabidopsis Stock Centre database (reference NASCARRAYS-642; http://affymetrix.arabidopsis.info/).

Bioinformatic analysis

Data analysis, including statistical analysis (ANOVA, P value <0.001),fold change values, plot representations, clustering and Venn dia-grams, was performed using ROSETTA RESOLVER software (RosettaBiosoftware, Seattle, WA, USA). Gene classification into functionalcategories was performed using BiNGO gene enrichment analysis(Maere et al., 2005). Microarray expression data of CK2 subunitswere obtained from the AtGenExpress project (http://www.arabid-opsis.org), data for the met1 mutant were obtained from Mathieuet al. (2007), and the list of transposable elements was obtainedfrom Slotkin et al. (2009).

CK2 activity assay, extraction of histones and

immunoblotting

CK2 kinase activity assays were performed as described by Moreno-Romero et al. (2008). The assays were done in triplicate, and back-ground signals (values obtained after adding 1 lM TBB to a reactiontest) were subtracted. Extraction of histones and immunoblottingwere performed as described by Friesner et al. (2005).

Comet assay

Comet assays were performed as described by Menke et al. (2001),with some modifications. Briefly, frozen material was chopped witha fresh razor blade on ice and under dim light in PBS containing10 mM EDTA. The suspensions of nuclei were filtered through80 lm mesh Sefar Nytal (Sefar AG, Heiden Switzerland) and mixedwith low-melting-point agarose (Bio-Rad) at 37�C, to a final con-centration of 0.38% w/v (alkali, A/A protocol) or 0.19% w/v (neutral,N/N protocol). Drops of 70 ll were loaded onto microscope slidespre-coated with 1% agarose, and covered with 20 · 20 mm coverslips, then incubated on ice for 5 min. In the A/A protocol, the slides



were incubated for 5 min in high-alkali buffer (0.3 M NaOH, 5 mM

EDTA, pH 13.5), prior to electrophoresis at 4�C in the same buffer(0.7 V cm)1), followed by a short neutralization step of 5 min in100 mM Tris/HCl, pH 7.5. In the N/N protocol, the slides were incu-bated for 1 h in high-salt buffer (2.5 M NaCl2, 10 mM Tris/HCl, pH 7.5,100 mM EDTA) on ice, followed by an equilibration step in 1· TBE(90 mM Tris/borate, 2 mM EDTA, pH 8.4) (3 · 5 min) prior to elec-trophoresis (1 V cm)1) at room temperature in TBE buffer. Theslides were then dehydrated in 75% v/v ethanol (5 min), followed by96% v/v ethanol (5 min), and air-dried. Dry agarose gels wererehydrated for 5 min in water and stained with 4¢,6-diamidino-2-phenylindole (DAPI) (1–5 lg ml)1). Images were captured using aLeica DRMB fluorescence microscope and quantified with COMET-

SCORE software, version 1.5 (Tritek Corp., Sumerduck, VA, USA). Atleast 100 nuclei were scored from two different comet gels for eachexperiment.

HR assay

Fourteen-day-old DGU.US or DU.GUS plantlets were treated witheither 10 lM TBB for 16 h or 10 lg ml)1 aphidicolin, or both, for 24 hin liquid medium, and then c-irradiated with 100 Gy and returned tothe growth chamber for the indicated times in each experiment.Aphidicolin was maintained in the growth medium for the wholetime, while TBB was removed after IR. The frequency of HR eventswas visualized as the number of blue tissue sectors after GUS his-tochemical staining. GUS activity was determined using a b-glucu-ronidase reporter gene staining kit (Sigma-Aldrich). Seedlings weremounted with 50% v/v glycerol and observed with a Leica DMRBmicroscope.

Micrococcal nuclease (MNase) assay

Nuclei from 7-day-old CK2mut plantlets (with or without dexa-methasone) were isolated by grinding plant material (2 g) in liquidnitrogen, and then resuspended in 0.25 M sucrose, 60 mM KCl,15 mM MgCl2, 1 mM CaCl2, 15 mM PIPES, pH 6.8, 0.8% Triton X-100and 1 mM PMSF. The homogenate was centrifuged for 20 min at10 000 g, and the pellet resuspended in 200–500 ll of 0.25 M

sucrose, 10 mM Tris/HCl pH 8.0, 10 mM MgCl2, 1% v/v Triton X-100,5 mM 2-mercaptoethanol and 1 mM PMSF. The suspension wasplaced on the top of an Eppendorf tube containing 1.7 M sucrose,10 mM Tris/HCl, pH 8.0, 10 mM MgCl2, 0.5% v/v Triton X-100, 5 mM

b-mercaptoethanol and 1 mM PMSF, and centrifuged at 4�C for 1 hat 12 000 g. The resulting pellet was resuspended in MNase buffer(0.3 M sucrose, 20 mM Tris/HCl, pH 7.5, 3 mM CaCl2), and the DNAconcentration was determined by measuring the absorbance at260 nm. Equal amounts of nuclei were digested, and the reactionwas stopped with 50 mM EDTA and 1% SDS. Samples were incu-bated with proteinase K overnight at 37�C, electrophoresed onagarose gels and visualized by gel staining with ethidium bromide.

ACKNOWLEDGEMENTS

We thank Dr H. Puchta (Botanisches Institut, Universitat Karlsruhe,Germany) for providing the DGU.US and DU.US reporter lines. Thiswork was supported by grants BFU2007-60569, BFU2010-15090 andConsolider Ingenio 2010 CSD2007-00036 from the Ministerio de Ed-ucacion y Ciencia, (Spain) and grants 2005SGR-00112 and 2009SGR-795 from the Generalitat de Catalunya, Catalunya, Spain. J.M.-R. andL.A. were recipients of fellowships from the Universitat Autonoma deBarcelona and the Ministerio de Educacion y Ciencia (Spain),respectively. We are indebted to the Arabidopsis InformationResource (TAIR) (http://arabidopsis.org) as an invaluable source ofdata, and to the Servei de Microscopia, Unitat Tecnica de Proteccio

Radioactiva and the Laboratori d’Analisi i Fotodocumentacio (Uni-versitat Autonoma de Barcelona, Spain) for technical support.

SUPPORTING INFORMATION

Additional Supporting Information may be found in the onlineversion of this article:Figure S1. Influence of auxin transport on IR hypersensitivity.Figure S2. Expression and activity of CK2 after genotoxic treat-ments.Figure S3. Validation of array data.Figure S4. DNA damage response and the cell cycle.Figure S5. DSB repair proficiency.Figure S6. MNase sensitivity.Figure S7. Transcript profiling of chromatin-related GO categoriesand heterochromatic silenced regions.Table S1. Global changes of gene expression in response to IR.Table S2. Primers used for real-time RT-PCR.Please note: As a service to our authors and readers, this journalprovides supporting information supplied by the authors. Suchmaterials are peer-reviewed and may be re-organized for onlinedelivery, but are not copy-edited or typeset. Technical supportissues arising from supporting information (other than missingfiles) should be addressed to the authors.

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