Plant Cell Rep DOI 10.1007/s00299-014-1580-0

ORIGINAL PAPER

Genome stability in the uvh6 mutant of Arabidopsis thaliana Andriy Bilichak • Youli Yao • Viktor Titov Andrey Golubov • Igor Kovalchuk



Received: 16 December 2013 / Revised: 21 January 2014 / Accepted: 30 January 2014 Ó Springer-Verlag Berlin Heidelberg 2014

Abstract Key message Plant XPD homolog UVH6 is the protein involved in the repair of strand breaks, and the excision repair and uvh6 mutant is not impaired in transgenerational increase in HRF. Abstract While analyzing the transgenerational response to stress in plants, we found that the promoter and gene body of Arabidopsis thaliana (Arabidopsis) XPD homolog UVH6 underwent hypomethylation and showed an increase in the level of transcript. Here, we analyzed the mutant of this gene, uvh6-1, by crossing it to two different reporter lines: one which allows for analysis of homologous recombination frequency (HRF) and another which makes it possible to analyze the frequency of point mutations. We observed that uvh6-1 plants exhibited lower rate of spontaneous homologous recombination but higher frequencies

of spontaneous point mutations. The analysis of strand breaks using ROPS and Comet assays showed that the mutant had a much higher level of strand breaks at noninduced conditions. Exposure to stresses such as UVC, heat, cold, flood and drought showed that the mutant was not impaired in an increase in somatic HRF. The analysis of spontaneous HRF in the progeny of control plants compared to that of the progeny of stressed plants demonstrated that uvh6-1 was mildly affected in response to temperature, UV and drought. Our data suggest that UVH6 may be involved in the repair of strand breaks and excision repair, but it is unlikely that UVH6 is required for transgenerational increase in HRF. Keywords XPD homolog  Homologous recombination frequency  Point mutation frequency  Abiotic stress  Excision repair capacity  Single- and double-strand break repair

Communicated by K. Chong.

Electronic supplementary material The online version of this article (doi:10.1007/s00299-014-1580-0) contains supplementary material, which is available to authorized users. A. Bilichak  Y. Yao  V. Titov  A. Golubov  I. Kovalchuk (&) Department of Biological Sciences, University of Lethbridge, University Drive 4401, Lethbridge, AB T1K 3M4, Canada e-mail: [email protected] A. Bilichak e-mail: [email protected] Y. Yao e-mail: [email protected] V. Titov e-mail: [email protected] A. Golubov e-mail: [email protected]

Background Plants can repair damaged DNA through a variety of mechanisms, including direct repair, base- and nucleotide excision repair (BER and NER), bypass repair, strand break repair [non-homologous end joining (NHEJ) and homologous recombination (HR)], mismatch repair (MMR) and several other mechanisms (Hays 2002). Among external stress factors that affect plant DNA is the UVC stress. Exposure to UVC mainly results in the generation of photoproducts such as cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidinone dimers (Hays 2002). Usually, plants are able to reverse these pyrimidine dimers via direct repair using the photolyase capacity to disrupt covalent bonds, thus removing a bridge between two

123

Plant Cell Rep

pyrimidines without removing the actual nucleotides. NER provides a backup for photolyase repair, especially in the dark (Kunz et al. 2006). The NER pathway seems to be conserved among eukaryotes. DNA lesions in plants are recognized by homologs of the human XPC/HR23B heterodimer (Liu et al. 2003). An incision of damaged nucleotides involves nicks in the 50 upstream region of the damage and is performed by plant homologs of the human XPF/ERCC1 heterodimer, AtRAD1/AtERCC1. The incision in the 30 region is performed by the AtXPG protein. Processing of damaged nucleotides involves DNA unwinding by two helicases, AtXPB and AtXPD, which are homologs of the human XPB and XPD proteins. In humans, mutations in the components of NER result in an extreme sensitivity to UVC light. Individuals with such mutations develop a disease such as xeroderma pigmentosum (XP); hence the genes in which mutations were found were named XPA, XPC, XPB, etc. Cells with mutations in the NER genes are not able to efficiently remove the pyrimidine dimers. In plants, Arabidopsis XPG and XPF mutants are also defective in the repair of UV photoproducts (Li et al. 2002; Liu et al. 2000), whereas atxpb1 plants are sensitive to alkylating agents (Costa et al. 2001). Analysis of atrad1 (atxpf) and atercc1 mutants did not show enhancement of chromosomal instability, although in the absence of telomerase (the attert mutant) these mutants exhibited a dramatic instability at the telomeres (Vannier et al. 2009). The AtXPD (UVH6) gene is of specific interest. Previously, we showed that the progeny of wild-type plants exposed to salt, UVC or heat exhibited transgenerational changes, namely, an increased frequency of spontaneous homologous recombination (HRF), a higher level of global genome methylation, differential changes in methylation at various gene-encoding loci and higher tolerance to stress (Boyko et al. 2010a, b. The AtXPD gene was among several DNA repair genes that was hypomethylated in the promoter regions (Boyko et al. 2010a). Hypomethylation in the promoter region of AtXPD appeared to correlate with permissive chromatin histone modifications and an increase in AtXPD expression (Boyko et al. 2010a). Thus, we hypothesized that hypomethylation of the promoter region and the increase in expression of DNA repair genes such as UVH6 could be one of the mechanisms of a transgenerational response to stress. Therefore, we decided to analyze the possible role of AtXPD in genome stability of somatic cells and in transgenerational responses to stress. An AtXPD mutant, known as uvh6, was first reported by Jenkins et al. (1995). It was identified among several other uvh mutants recovered from the EMS-mutagenized population based on UVC sensitivity (Jenkins et al. 1995). It

123

was shown that the uvh6 mutant was mildly sensitive to UVC and not sensitive to gamma rays. Later on, Jenkins et al. (1997) reported the uvh6 mutant to be heat stress sensitive. They also demonstrated that heat shock protein 21 (HSP21) accumulated to a higher level and required a less severe heat stress exposure for accumulation as compared to wild-type plants (Jenkins et al. 1997). Finally, Liu et al. (2003) reported two uvh6 mutants, the uvh6 mutant designated by the authors as uvh6-1 and a new uvh6-2 mutant (Liu et al. 2003). Whereas the uvh6-1 mutant was shown to have the amino acid substitution of Glu for Lys at codon position 554, the uvh6-2 mutant was a T-DNA knockout mutant, lethal at homozygosity. The authors were able to confirm the UVC-sensitive phenotype of uvh6-1 and showed that the mutant can be complemented by overexpression of AtXPD genomic DNA. They also showed that the repair of (6-4) photoproducts was moderately defective in the mutant. Based on the aforementioned observations and on the fact that amino acid substitution in the uvh6-1 mutant occurred between motifs III and IV required for helicase activity, the authors suggested that the Arabidopsis homolog AtXPD/UVH6 was highly likely to function in NER as a repair helicase (Liu et al. 2003). Vonarx et al. (2006) showed the interaction between Arabidopsis, a homolog of the essential interacting human/ yeast TFIIH components p44/Ssl1, AtGTF2H2 and AtXPD. AtXPD also interacted with Scl1, a component of yeast TFIIH, in a two-hybrid assay (Vonarx et al. 2006). This suggests that AtXPD may also be involved in the regulation of transcription. The work of Larkindale et al. (2005) confirmed that uvh6 mutant plants exhibited heat sensitivity and also showed the inability of these mutants to establish basal and heat-induced thermotolerance (Larkindale et al. 2005). The authors drew a parallel between the lack of response to certain hormones in mammalian cells deficient in XPD and the inability of Arabidopsis uvh6 to establish thermotolerance. They suggested that this was due to the fact that AtXPD might have a function in transcription regulation. Could AtXPD serve a dual function as a DNA repair enzyme and a transcription factor? This remains to be shown. Both the yeast homolog RAD3 and the homolog of human XPD have a dual role in NER and transcription initiation (de Boer and Hoeijmakers 2000; Prakash and Prakash 2000). It was suggested that XPD (RAD3) is a part of the initiation factor TFIIH that facilitates the initiation of transcription by RNA polymerase II. In fact, it was proposed that mutations in human XPD cause developmental abnormalities due to defects in transcription (de Boer and Hoeijmakers 2000). Mutations in this single gene lead to three different genetic diseases, namely, xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy (de Boer and Hoeijmakers 2000; Lehmann 2001).

Plant Cell Rep

Although these disorders are commonly characterized by UV light sensitivity and repair deficiencies, they are distinguished by additional distinct phenotypes. Presumably, these additional phenotypes are mainly due to problems with transcription rather than DNA repair capacity (de Boer and Hoeijmakers 2000; Lehmann 2001). In this respect, the phenotype of uvh6 mutants resembles the phenotype of patients with the aforementioned human diseases. The uvh6-1 mutant that carries a mutation in the helicase domain exhibits UVC sensitivity and a reduced capacity to remove (6-4) photoproducts. It is also heat stress sensitive, whereas the null uvh6-2 mutant is lethal. Recently, Hall et al. (2009) showed that uvh6-1 plants were also extremely sensitive to cold (Hall et al. 2009). The authors showed that cold sensitivity of uvh6-1 plants is in part due to the inability to activate expression of two coldstress genes, Cor6.6 and Cor15a, suggesting that UVH6 possibly functions as transcription factor regulating the expression of Cor6.6 and Cor15a genes. Thus, it appears that the uvh6-1 mutant does not have a visibly different phenotype unless it is exposed to stress. Here, we report that the uvh6-1 mutant has an increased level of strand breaks, a lower capacity to repair strand breaks, a nearly similar excision repair capacity, a lower frequency of spontaneous recombination, a higher frequency of spontaneous mutations and higher expression of several DNA repair enzymes. The analysis of changes in the progeny of stressed plants shows that the mutant is only mildly affected by transgenerational changes in recombination frequency.

illumination (100 lM m-2 s-1). Exposure to flood stress was done by watering plants every day, making sure that the pots were standing in water all the time. Drought conditions were created by stopping watering between days 7 and 30 post-germination. Watering was resumed on day 30 to obtain progeny seeds. To analyze the influence of heat stress, plants were germinated in soil and exposed to 42 °C for 2 h/day for 1 week starting at 7 days post-germination. The effect of cold stress was analyzed when plants were exposed to 4 °C for 12 h during the night for 1 week starting at 7 days post-germination. Exposure to UVC was done at 14 days post-germination using germicidal lamps; plants received 0.6 or 1.2 J m-2 as a single acute dose (0.01 J m-2 s-1). Irradiations were done at the same time, at 10–11 a.m. and plants were grown at normal conditions (see above) after exposure. For salt stress, plants were germinated and grown on sterile medium supplemented with 0, 25 or 75 mM NaCl. None of these conditions resulted in changes in plant appearance. In all cases, the plants were analyzed for point mutations and homologous recombination frequencies at 21 days post-germination. All experiments were repeated three times. For the analysis of transgenerational effects, ten plants from each line per each treatment were allowed to set seeds. To analyze spontaneous non-induced changes in genome stability in the progeny, the seeds of stressed and non-stressed plants were germinated in soil and grown for 3 weeks at 22 °C during a 12-h day period and at 18 °C during a 12-h night period under illumination (100 lM m-2 s-1) and the normal watering regime; and HRF was measured at day 21 post-germination.

Materials and methods

The histochemical staining procedure for the analysis of point mutations

Plant lines and growing conditions Two different transgenic Arabidopsis thaliana lines were used in the experiments. The transgenic line #166-14 carried the b-glucuronidase (GUS)-based substrate in its genome for the analysis of T/A ? G/C reversions (Kovalchuk et al. 2000a). The transgenic line #15d8 carried the luciferase-based substrate in its genome for the analysis of homologous recombination frequency (Boyko et al. 2006c). To analyze point mutation events and recombination events in the uvh6-1 mutant plants (Col-0 background), individual mutant plants were crossed either with line #166-14 (C24 cultivar) or line #15d8 (Col-0 background). Plants homozygous for the recombination/mutation substrate and mutations of the UVH6 gene (as determined by gene-specific or transgene-specific PCRs) were used for further experiments. All plants were grown in soil at 22 °C during a 12-h day period and at 18 °C during a 12-h night period under

The histochemical staining was done at 7 days post-treatment as described before (Ilnytskyy et al. 2004). For the analysis, the whole 3-week-old plants without roots were used. For destructive staining, the plants were vacuum infiltrated (2 9 10 min) in 10 ml of a sterile staining buffer. The buffer was prepared by adding 100 mg of the 5-bromo-4-chloro-3-indolyl glucuronide (X-gluc) substrate (Jersey Labs Inc., USA) to 300 ml of 100 mM phosphate buffer (pH 7.0) and supplemented with 0.05 % NaN3 (for decreasing the potential bacterial growth) and 0.1 % Triton X-100 (for improving the penetration of the substrate into cells). Afterward, the plants were incubated at 37 °C during 48 h and bleached with ethanol multiple times until a sufficient tissue transparency was reached. The non-transgenic plants were used as a negative control. Point mutation frequency was calculated by scoring the number of events (sectors of blue) and relating it to the total number of plants scored (Kovalchuk et al. 2000a). Since the

123

Plant Cell Rep

frequency of occurrence of these events is low, a population of 300–400 plants per experimental point was used. The average point mutation frequency was calculated at 3 weeks post-germination from three independent experiments.

with three measurements/views made. The average with SD was calculated for both lines.

Analysis of HRF using a luciferase camera

The quantification of 30 OH DNA breaks was performed using the random oligonucleotide-primed synthesis (ROPS) assay as described before (Yao et al. 2011). Wildtype and uhv6-1 plants were irradiated with 1.2 J m-2 of UVC, and half of the plants were kept in the dark, whereas the other half was kept under light conditions. Tissues were collected at 2 h post-irradiation. Total genomic DNA was also prepared as described (Yao et al. 2011). The assay is based on the ability of the Klenow fragment polymerase (New England Biolabs) to initiate random oligonucleotideprimed synthesis from the re-annealed 30 OH ends of single-stranded (ss) DNA. After a denaturation–reassociation step, the ssDNA serves as its own primer by randomly reassociating itself to other ssDNA molecules. Under strictly defined reaction conditions, the incorporation of [3H]-dCPT into a newly synthesized DNA will be proportional to the initial number of 30 OH ends (breaks). The experiment was performed three times, with two independent measurements per experiment.

The frequency of homologous recombination in 15d8/ uvh6-1 homozygous Arabidopsis plants carrying the luciferase reporter was analyzed using a luciferase CCD camera by scoring bright sectors on a dark background after spraying with luciferin (Boyko et al. 2006c). HRF was calculated by relating the number of recombination events in the entire population of tested plants to the total number of plants scored. The average HRF was calculated at 3 weeks post-germination using three to five independent experiments. In each experiment, three different pots were used (20–25 plants in each of them). Calculation of the number of genomes and mutation and recombination rates Total DNA of 3-week-old wt and uvh6-1 plants was isolated from whole plants without roots using a Nucleon Phytopure plant DNA extraction kit (Amersham Life Science, Piscataway, New Jersey, USA) and quantified by Nanodrop. The yield of total DNA was divided by the average DNA content (0.16 pg) of an A. thaliana cell to get an estimate of the number of genomes present (Swoboda et al. 1993). Four independent DNA preparations from four groups of 10–15 plantlets each were done. The mutation and recombination rates were calculated by relating PMF or HRF to the average number of genomes present in wt or uvh6-1 plants. Analysis of mesophyll cell density and leaf thickness The fully extended leaves were fixed by FAA at 18 days after germination. The view pictures were taken at the virtual middle line between the edge and the central vascular vein (the midrib) of the leaf. Three views were photographed using Zeiss AX10. All measurements were taken under 109 magnification, and 22 pictures/views were enumerated for each line. The cells were enumerated using analysis software. Leaf thickness was measured using paraffin sections of fixed leaves. Section slides were stained by Safranin O and Orange G. Leaf thickness was measured at the same location that was used for counting mesophyll cells. All measurements were taken under 109 magnification; for measurements, we used 32 views for 15D8 and 27 for uvh6,

123

Analysis of double-strand break (DSB) and single-strand break (SSB) levels

Real-time PCR analysis Approximately 80 mg of plant tissue was ground in liquid nitrogen and transferred to a chilled 1.5 mL Eppendorf tube; 160 lL TRIzol reagent (Invitrogen) was added. The remainder of the extraction was performed as per the manufacturer’s protocol. RNA quantity and quality were measured using a spectrophotometer (Ultraspec 1100 pro) in 20 mM Tris pH 7.5 in RNase-free double-distilled water. cDNA was then prepared from total RNA using the RevertAID H first-strand cDNA synthesis kit (Fermentas). Real-time quantitative PCR (RT-qPCR) was performed using SsoFast EvaGreen Supermix (Bio-Rad). cDNAs were amplified under the following conditions: (a) 98 °C for 3 min for one cycle; (ii) 98 °C for 5 s, 54.3 °C for 5 s and 65–95 °C for 5 s, with an increment of 0.5 °C for 40 cycles. Primers (see Table S1) for RT-qPCR were designed using Primer3 program. For every set of primers, annealing temperature optimization, melting curve analysis and gel analysis of amplicon were performed. To evaluate the efficiency of PCR, the standard curve was established using a series of cDNA dilutions. The expression of aforementioned genes was related to the expression of RCE1 (RUBconjugating enzyme 1). The expression of RCE1 did not change upon exposure to UVC (data are not shown). Conditions of RCE1 amplification were as follows: (a) 98 °C for 3 min for one cycle; (b) 98 °C for 5 s,

Plant Cell Rep

45.0 °C for 5 s, 65–95 °C for 5 s, with an increment of 0.5 °C for 46 cycles. The average of 4 reactions (2 dilutions per each of two RNA preparations) was obtained and the relative ratios were calculated. The statistical significance of the experiment was confirmed by performing a Student’s t test (two-tailed paired or non-paired). Protoplast isolation for the comet assay Protoplast isolation was done using the Tape-Arabidopsis Sandwich method as described previously (Wu et al. 2009). Healthy leaves (length 2–3 cm) were collected from UVCirradiated (see above) and control 3-week-old plants grown in soil at 22 °C under 12-h day/12-h night conditions and illumination at 100 lM m-2 s-1. Tissues were collected 2 h after irradiation. The enzyme solution that was used for protoplast isolation containing 20 mM MES (pH 5.7), 0.4 M mannitol, 20 mM KCl, 1.5 % (wt/vol) cellulase R10 (Yakult Pharmaceutical Ind. Co., Ltd.) and 0.4 % (wt/vol) macerozyme R10 (Yakult Pharmaceutical Ind. Co., Ltd.) was warmed at 55 °C for 10 min to inactivate DNase and proteases and enhance enzyme solubility (Yoo et al. 2007). Then, the solution was cooled to room temperature, and CaCl2 and BSA were added to a final concentration of 10 mM and 0.1 %, respectively. The enzyme solution was prepared fresh every time. To expose mesophyll cells to the enzyme solution, the lower epidermal surface was peeled away using a Magic tape (3 M, St. Paul, MN). The peeled leaves (4 to 6 leaves) adhering to the Time tape (Time Med, Burr Ridge, IL) were transferred to a Petri dish containing 10 mL of the enzyme solution. The leaves were gently shaken (40 rpm) for 60 min in the dark until protoplasts were released into the solution. Then the protoplasts were collected by centrifugation (100 g, 3 min, 4 °C), washed twice with 25 mL of a prechilled W5 solution (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 5 mM glucose and 2 mM MES, pH 5.7) and resuspended to a final concentration of 2–5 9 105 cells/mL. The resulting protoplast solution was kept on ice until further processing. The comet assay DSB levels were measured according to the protocol described for the neutral comet assay (Kozak et al. 2009). 50 ll of the protoplast suspension was mixed with 200 ll of molten 0.7 % low-melting-point agarose (15,517-022, Invitrogen) at 40 °C, and the resulting solution was immediately pipetted onto a fully frosted, agarose-coated microscope slide (12-544-5CY, Fisher Scientific). The slides were covered with 22 9 50 mm coverslips and chilled on ice for 1 min to solidify the agarose. After the removal of the coverslips, the slides were immersed in a lysing solution (2.5 M NaCl, 10 mM Tris–HCl, 0.1 M

EDTA, 1 % N-lauroylsarcosinate, pH 7.6) and incubated on ice for 1 h to dissolve cellular membranes and remove attached proteins. The slides were equilibrated two times for 5 min in TBE buffer. Then the slides were subjected to electrophoresis at 1 V/cm for 5 min. After electrophoresis, the comet slides were dehydrated for 5 min in 70 % EtOH, 5 min in 96 % EtOH and air dried. Then they were stained with SYBR Gold Nucleic Acid Gel Stain for 20 min and viewed in epifluorescence with a Zeiss Observer Z1 microscope using the Stingray CCD camera (Allied Vision Technologies). Comets were evaluated with the Comet Assay IV software system (Perceptive Instruments Ltd.) using single click automatic scoring. The Olive tale moment values were used to measure DNA damage. The experiments consisted of two independent biological replicas, each including two technical repeats. Totally, at least 100 comets were analyzed per experimental group. Statistical analysis The experiments were repeated three times and the mean values with SD were calculated. The statistical significance of the results was confirmed by performing a Student’s t test. Statistical analyses were performed using Microcal Origin 6.0 software.

Results Analysis of the frequency of spontaneous and stressinduced point mutations Since uvh6-1 plants are impaired in the XPD homolog, we hypothesized that they are impaired in NER. Therefore, it is possible that point mutation frequency in uvh6-1 plants is higher than in the wild-type plants. To test this hypothesis, we crossed uvh6-1 plants with plants from Line #166-14 carrying a substrate for the analysis of T/A ? G/C reversions in their genome (Kovalchuk et al. 2000a). These plants were in past used for the analysis of point mutations upon exposure to different stresses (Kovalchuk et al. 2000b; Van der Auwera et al. 2008; Yao et al. 2011; Yao and Kovalchuk 2011). The analysis demonstrated that the spontaneous frequency of T/A ? G/C reversions in uvh6-1 plants was nearly threefold higher than that in wild-type (wt) plants (Fig. 1a; Table S2). Exposure to NaCl showed that mutation frequency in uvh6-1 plants did not increase in response to this stress, whereas it did increase in wild-type plants. In wild-type plants, exposure to 25 and 75 mM NaCl increased the mutation frequency by 1.50- and 1.75fold, respectively. In contrast, exposure of uvh6-1 plants to 25 and 75 mM NaCl did not change the mutation frequency.

123

Plant Cell Rep

0.16 0.14 0.12

4.45 **

wt uvh6

the spontaneous mutation rate was nearly 30 % higher as compared to wt plants. In the mutant plant, the mutation rate did not change in response to salt but increased in response to UVC.

0.1 0.08

Analysis of mesophyll cell density and leaf thickness

** 2.58

0.04

**

*

1.18

1.5

3.58

* 1.75

0.74

0.02 0

B

6E-09

Mutation rate, events per genome

ct

5E-09

25 mM

75 mM

UV

**

wt uvh6

4E-09

** 3E-09 2E-09

* *

1E-09 0 ct

25 mM

75 mM

UV

Stress type

Fig. 1 Point mutation frequency and mutation rate in wild-type and uvh6-1 plants. a Point mutation frequency was measured in 3-weekold uvh6-1 (uvh6) and wild-type (wt) plants exposed to salt or UVC stresses. The data are shown as the average number (with SD) of events per single plant as calculated in the population of 300–400 plants per each experimental group. The numbers over the bars show the fold increase of PMF in stressed plants. The asterisks show a significant difference between stressed and non-stressed plants; one asterisk if p \ 0.05 and two asterisks if p \ 0.01. b Point mutation rate in wt and uvh6-1 plants. The asterisk shows significant difference between wt and mutant plants (p \ 0.05)

Since the nucleotide at the 50 position of the GUS gene is also a T nucleotide, it is possible to analyze point mutations generated as a result of mistakes upon repair of UVC-induced pyrimidine dimers (Kovalchuk et al. 2000a). We found that exposure to UVC resulted in a 3.58-fold increase in the point mutation frequency in wild-type plants and a 4.45-fold increase in uvh6-1 plants (Fig. 1a). Analysis of mutation rate Mutant and wild-type plants may differ in the number of genomes in a plant of a given age. Thus, the mutation rate is calculated by relating the mutation frequency to the number of genomes present in the plant (Boyko et al. 2006c). We analyzed the number of genomes in 3-weekold wild-type plants and uvh6-1 plants and found that wt plants had 1.37E?07±0.10E?07 genomes, whereas uvh61 plants had 2.75E?07±0.11E?07 genomes (Fig. 1b). The mutation rate analysis thus showed that in uvh6-1 plants,

123

It was surprising to find that mutant plants had nearly twofold more genomes than wt plants. A higher number of genomes may suggest that mutant plants have more frequent cell divisions or more frequently undergo endoreduplication, a process of genome replications without cell divisions. To test whether uvh6 plants potentially have more cells, we analyzed the mesophyll cell density and leaf thickness. In uvh6 mutants, we have found an increase in leaf thickness of 15.7 % and in the number of mesophyll cells of 6.8 %, although both differences were not significant (Fig. S1A-B). It is unlikely that these differences in leaf number/leaf thickness account for differences observed in the amount of genomic DNA extracted from uhv6 plants (Fig. S1E). Thus, we cannot exclude that uvh6 plants may also have a higher frequency of endoreduplication. Analysis of homologous recombination frequency To analyze the homologous recombination frequency (HRF) in uvh6-1 Arabidopsis plants, we crossed the mutants to the Arabidopsis plants from Line #15d8 that carried the homologous recombination substrate in the genome. This recombination substrate allowed us to measure the recombination frequency between sister chromatids and homologous chromosomes. Earlier, these plants were used for the analysis of genome stability of various mutants (Yao et al. 2010) and for the analysis of the influence of various genotoxic agents on HRF (Boyko et al. 5 wt

uvh6

4 3 2 1 0 1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113 117

0.06

Number of plants

PMF. events per plant

A

Number of spots

Fig. 2 Plants impaired in UVH6 have lower frequencies of homologous recombination. Events of homologous recombination were calculated in the population of *90 wild-type (wt) and uvh6-1 (uvh6) plants grown under normal non-induced conditions (see Materials and methods). The figure shows the distribution of HR events from 0 to 120 spots per plant

Plant Cell Rep

2006a, b, 2010a; Yao et al. 2011; Yao and Kovalchuk 2011). The analysis of HRF showed that uvh6-1 plants had an average of 27.6 spots per plant, whereas wild-type plants had an average of 39.7 spots per plant. As shown in Fig. 2, the lower frequency of homologous recombination (HRF) is primarily due to the less frequent occurrence of plants with a large number of recombination events. This analysis indicated that uvh6-1 plants are partially deficient in HR repair. The analysis of the recombination rate (RR) and the number of recombination events per single cell genome showed that wt plants had a significantly higher (p \ 0.05) number of events per single cell genome as compared to uhv6-1 plants, 2.91E-06 versus 1.00E-06 events, respectively. To test whether uvh6-1 mutant plants are also impaired in response to stress, we exposed mutant and wild-type plants to UVC (0.6 and 1.2 J/m-2) (Fig. S2), heat (37 and

HRF, events per plant

A

300 250

uvh6

** 4.76

200

2.89

*

150

3.89

*

100

3.46

3.20

*

*

1.83 2.54 * 1.41 1.46 * 1.49 1.03 *

50

0.690.93

0 ct

B RR, events per genome

wt

6.58

UV0.6 UV1.2

1.4E-05

4.70

1.2E-05

**

37C

50C

4C

DR

wt uvh6

3.40

1.0E-05

* 2.06

8.0E-06 5.45

6.0E-06

*

4.0E-06 2.0E-06

FL

4.84

4.48

1.31 3.55 * 1.00 2.09 2.04 0.73

*

* 0.50 1.31

*

*

0.0E+00 ct

UV0.6 UV1.2

37C

50C

4C

FL

DR

Stress type

Fig. 3 HRF and recombination rate in uvh6-1 and wild-type plants exposed to stress. a HRF was calculated in plants exposed to UVC (0.6 and 1.2 J/m2, UV0.6 and UV1.2, respectively), heat (37 and 50 °C), cold (4 °C), flood (FL) and drought (DR). The data are shown as the average (of three independent experiments with SD) recombination frequency. The numbers over the bars show the fold increase of HRF in stressed plants. The asterisks show a significant difference in HRF between exposed and non-exposed plants; one asterisk if p \ 0.05 and two asterisks if p \ 0.01. b Recombination rate in wt and uvh6-1 plants. The asterisk shows significant difference between wt and mutant plants as well as between stressed and non-stressed plants (p \ 0.05)

50 °C), cold (4 °C), flood and drought. The analysis showed that HRF or RR increased in response to UVC in a similar manner in wt and mutant plants (Fig. 3a, b). We found that wt plants responded to UVC exposure with a higher increase in HRF, but with a lower increase in RR (mainly due to the fact that wt plants had fewer genomes per plant as compared to uvh6 mutant plants) (Fig. 3a, b). Also, in both wt and mutant plants, HRF and RR did not significantly change in response to cold and drought. Changes in HRF and RR in response to high temperature and flood varied, with mutants having a significant increase in all cases, whereas wt plants demonstrated a significant increase only in HRF in response to 50 °C (Fig. 3a, b). Analysis of the level of DNA strand breaks in uvh6-1 and wild-type plants In the past, uvh6-1 plants were shown to be defective in the removal of (6-4) photoproducts (Liu et al. 2003). Here, we show that these plants have an increased frequency of point mutations and a decreased frequency of homologous recombination. Lower frequency of HR repair could result in higher level of strand breaks accumulating in uvh6-1 plants. To test this hypothesis, we performed two different assays, the random oligonucleotide-primed synthesis (ROPS) assay (Golubov et al. 2010) and the neutral comet assay (Collins 2004). Whereas the ROPS assay shows the presence of 30 OH DNA breaks measured by the rate of incorporation of radioactively labeled nucleotides initiated by the Klenow fragment polymerase, the neutral comet assay reflects DSBs measured by the tail moment of the comet formed during gel electrophoresis (Collins 2004). The ROPS assay displayed a 4.7-fold increase (p \ 0.001) in the incorporation of radioactive dNTPs in the non-exposed mutant versus wild-type plants (Fig. 4a). To analyze whether strand breaks appear as a result of either direct damage by UVC or the appearance of transient strand breaks upon repair of (6-4) photoproducts, we placed half of UVC-irradiated plants in the dark immediately after exposure, whereas the other half remained under light conditions. We found that UVC exposure resulted in a significant increase in the level of strand breaks in wt, but not in mutant plants. Placing plants in the dark resulted in a further increase in strand breaks in wt but not in mutant plants, suggesting that (6-4) photoproducts are repaired by nucleotide excision repair in wt plants (and therefore transient strand breaks accumulate); in contrast, lack of further increase in the level of strand breaks in the mutant plants placed in the dark suggests that they are deficient in NER (Fig. 4a). The comet assay showed a 1.9-fold increase in the level of spontaneous DSBs (p \ 0.05) in uvh6-1

123

Plant Cell Rep

*

1.05

1.12

14

***

1.2E+05 1.0E+05 8.0E+04

1.69

*

6.0E+04

**

1.38

4.0E+04

B DSBs, tail moment

SSBs and DSBs level

A 1.4E+05

*

2.0E+04

10

C

8 6 4 2 0

0.0E+00

UVC dark

*

12

wt

wt

wt

-

+ -

+ +

wt

uvh6

-

uvh6

wt

uvh6

+ -

wt+UV

wt+UV

uvh6

uvh6+UV

+ +

uvh6

uvh6 +UV

Fig. 4 uvh6-1 plants have a higher level of strand breaks than wildtype plants. a Single-strand breaks (SSBs) and double-strand breaks (DSBs) were measured in uvh6-1 and wild-type plants using ROPS. The Y-axis shows the level of strand breaks as the average (of two independent experiments, each with two technical repeats with SD) amount of 3H incorporation (dpm/lg). The asterisks show a significant difference in strand breaks level between uvh6-1 and wild-type plants (p \ 0.001) as well as between UVC-exposed and non-exposed plants (one—p \ 0.05 and two—p \ 0.01). Numbers

over the bar show the fold difference in strand breaks between UVCexposed and non-exposed or UVC-exposed and incubated in the dark and just UVC-exposed plants. b DSBs are reflected as the mean (of two independent experiments, each with two technical repeats with SD) tail moment of comets measured using the comet assay. The asterisk shows a significant difference in strand breaks level between uvh6-1 and wild-type plants (p \ 0.05). C. The representative images of comets and tails of non-exposed and exposed wild-type and uvh6-1 plants. See Materials and methods for details

mutant plants (Fig. 4b, c). The difference is that the ROPS assay can measure all existing repair intermediates that lead to single-strand breaks (SSBs) and DSBs, whereas the comet assay primarily measures DSBs. Exposure to UVC resulted in a 1.3-fold increase in DSBs in the wild-type plants and no change in the mutant plants (Fig. 4b). These experiments showed that the mutant plants had high spontaneous levels of SSBs and DSBs, but the level of DSB was not substantially induced by UVC exposure.

significantly higher, whereas the expression level of KU70 was lower in uvh6-1 plants as compared to wild-type plants (Fig. 5). Exposure to UVC (1.2 J/m-2) resulted in an increase in the expression level of POL d and RAD51 genes in both wild-type and mutant plants. In contrast, a decrease in the expression level of POL a in the UVC-treated wildtype plants (p \ 0.05) was not observed in the mutant plants (Fig. 5a). Finally, the expression of KU70 did not change in either plant line exposed to UVC (Fig. 5c). The deficiency in NER may be compensated by other repair pathways such as bypass and translesion synthesis. We tested the expression of polymerases AtREV1, AtREV3, POL g, POL j and found that all these genes had lower expression as compared to wt plants (Fig. 6). The expression of AtREV1 was induced by UVC, whereas the expression of other three enzymes was downregulated.

Analysis of expression of repair genes in uvh6-1 and wild-type plants Since uvh6-1 plants had a higher level of spontaneous strand breaks as well as changes in HRF, mutation frequency and repair capacity, we hypothesized that these plants would also have changes in the expression of repair genes and genes that are in general involved in DNA metabolism. To test this assumption, we checked the expression of POL a, the replicative polymerase with the primase activity, POL d, the main replicative polymerase, KU70, the enzyme involved in NHEJ, and RAD51, the enzyme which is essential for homologous recombination repair. The real-time PCR analysis showed that the expression level of POL a and POL d genes was

123

Analysis of transgenerational changes in HRF in uvh61 and wild-type plants Finally, to examine whether UVH6 may play any role in transgenerational response to stress, we propagated seeds of stress-exposed and control wild-type and uvh6-1 plants to the next generation. The analysis of spontaneous HRF in the progeny of exposed plants versus the progeny of

1.40 1.20

wt uvh6

C *

Expression, arbitrary units

A Expression, arbitrary units

Plant Cell Rep

*

1.00 0.80

*

0.60 0.40 0.20 0.00 ct 1.80 1.60 1.40 1.20 1.00 0.80 0.60 0.40 0.20 0.00

ct

D wt uvh6

* * * **

Expression, arbitrary units

AtREV1

wt

*

2.5

1.00 0.50

2

B

wt

AtREV3

uvh6

2

*

1.5

*

1

3 2.5

**

*

1.5

UVC

type and uvh6-1 plants. The asterisks show a significant difference between wt and mutant and between UVC-exposed and non-exposed (p \ 0.05)

uvh6

0.5

**

**

1 0.5 0

ct

Expression, arbitrary units

*

ct

0

C

*

RAD51 *

1.50

UVC

Fig. 5 uvh6-1 plants have higher expression levels of Pol d, Ku70 and RAD51 genes The steady-state level of mRNA of Pol a (a), Pol d (b), Ku70 (c) and RAD51 (d) genes was analyzed in 3-week-old wild-

3

wt uvh6

UVC

0.00 ct

A

2.00

wt uvh6

KU70

*

UVC

Expression, arbitrary units

Expression, arbitrary units

B

2.20 2.00 1.80 1.60 1.40 1.20 1.00 0.80 0.60 0.40 0.20 0.00

1.6

UVC wt

POL

1.4

ct

uvh6

1.2

*

1 0.8

3

wt

POL

2.5

uvh6

*

2

**

0.6

D

UVC

1.5

**

1

*

0.4 0.5

0.2 0

0

ct

UVC

ct

UVC

Fig. 6 uvh6-1 plants have lower expression levels of AtREV1, AtREV3, POL j and POL g genes. The steady-state level of mRNA of AtREV1 (a), AtREV3 (b), POL j (c) and POL g (d) genes was analyzed in 3-week-old wild-type and uvh6-1 plants. The asterisks

show a significant difference between wt and mutant and between UVC-exposed and non-exposed; one asterisk if p \ 0.05, two asterisks if p \ 0.01

stressed plants demonstrated that both wild-type and mutant plants responded to stresses with a transgenerational increase in HRF (Fig. 7, Fig. S3). However, the increase in HRF in response to UVC, heat, cold and drought stress was lower in uvh6-1 plants as compared to wild-type plants. This experiment suggests that uvh6-1 plants are mildly affected in their ability to establish transgenerational increase in HRF in response to stress.

Discussion Here, we tested genome stability of uvh6-1, a homolog of human XPD, and found these plants to have a lower rate of spontaneous homologous recombination, higher frequency of spontaneous point mutations, higher levels of DSBs and different levels of expression of enzymes involved in DNA metabolism. The uvh61-1 plants responded to UVC stress

123

Plant Cell Rep

*

HRF, fold to control

5

***

*

wt

4

**

uvh6

** **

* 3

*

**

**

**

** 2

*

*

1

fact that uvh6-1 plants have lower expression levels of KU70 and translesion synthesis polymerases may suggest against more frequent involvement of NHEJ and bypass repair in the processing of pyrimidine dimers in uvh6-1 plants exposed to UVC. Although no information exists about the frequency of point mutations in other NER mutant plants, yeast cells deficient in RAD3 exhibit an increased frequency of spontaneous mutagenesis, including point mutations (Malone and Hoekstra 1984).

0 ct

UV0.6

50C

4C

FL

DR

Stress type Fig. 7 The homologous recombination frequency in the progeny of stressed wild-type and uvh6-1 plants The frequency of homologous recombination was calculated in the 3-week-old progeny of plants exposed to UVC (0.6 J/m2; UV0.6), heat (50 °C), cold (4 °C), flood (FL) and drought (DR). The data are shown as the average fold increase (of three independent experiments with SD) in HRF in nonexposed plants. The asterisks show a significant difference in HRF between both exposed and non-exposed plants and uvh6-1 and wildtype plants; one asterisk if p \ 0.05, two asterisks if p \ 0.01 and three asterisks if p \ 0.001

with a lower increase in HRF in somatic tissue and in the progeny. uvh6-1 plants have a higher frequency of point mutations uvh6-1 plants are sensitive to UVC and heat and are partially compromised in the removal of (6-4) photoproducts (Liu et al. 2003). The higher frequency of spontaneous point mutations in uvh6-1 plants observed in this work could be due to the deficiency in NER. Exposure to UVC resulted in an even higher PMF. It suggests that additional (6-4) photoproducts are less efficiently taken care of in uvh6-1mutant plants, which also leads to a higher frequency of T/A ? G/C mutations. We tested this hypothesis by incubating UVC-exposed plants in the dark for 2 h immediately after exposure. Since photolyase repair requires light, these photoproducts cannot be removed via this repair pathway (Ries et al. 2000). Instead, NER repairs these lesions. Repair of (6-4) photoproducts results in the formation of transient strand breaks. The analysis of the level of strand breaks in UVC-exposed plants incubated in the dark showed a 67 % increase in the number of breaks in wt, but only a slight 5 % increase in mutant plants. This further suggests that uvh6-1 plants are impaired in NER. An increased number of mutations could also be explained by the utilization of bypass repair or NHEJ repair, with both being highly error prone. We do not have any experimental proof that this is the case. Moreover, the

123

uvh6-1 plants exhibit higher levels of DSBs A higher level of strand breaks observed in uvh6-1plants is not surprising. The lack of proper processing of damaged nucleotides due to mutation in UVH6 possibly results in the accumulation of strand break intermediates. These intermediates are mainly SSBs rather than DSBs. Indeed, the difference between the levels of SSBs inuvh6-1 and wild-type plants was much larger than that between their levels of DSBs, suggesting that NER intermediates do accumulate in mutant plants. In humans, the correlation analysis showed a higher incidence of chromatid breaks in cells with XPD mutations (Kazimirova et al. 2009). Also, human fibroblasts deficient in yet another helicase involved in NER and XPB exhibited a higher level of c-H2AX upon exposure to UVC, suggesting that the increased level of strand breaks in these mutants is relatively common (Oh et al. 2011). In contrast, our analysis of Arabidopsis thaliana shows that mutant plants have higher levels of spontaneous breaks, but these levels do not change upon UVC exposure. It can be suggested that some compensatory mechanisms exist to make up for an increase in the number of UVC-induced breaks. uvh6-1 plants have a lower rate of homologous recombination Our experiments showed that uvh6-1 plants had a significantly lower RR and there were fewer plants with high number of recombination events. It is not clear why there were fewer plants with a large number of recombination events among the uvh6-1 plants. Homologous recombination requires strand breaks to occur. The breaks are produced internally as a result of oxidative damage caused by cell metabolites or as a result of malfunctions during replication/transcription (Tuteja et al. 2001). External agents capable of breaking DNA are primarily represented by ionizing radiation, although some chemicals and even UVC are able to induce strand breaks (Boyko et al. 2006a; Ilnytskyy et al. 2005; Kovalchuk et al. 2000b; Puchta 2005). At the same time, the repair of various damages to bases or nucleotides results in the generation of transient

Plant Cell Rep

strand breaks. For example, the processing of pyrimidine dimers by NER generates transient strand breaks that upon malfunction of NER can be processed by NHEJ/HR repair (Hays 2002). In the case of uvh6-1plants, it would be logical to assume that inefficient NER would lead to more frequent NHEJ/HR which would result in a higher frequency of homologous recombination. In fact, this is what happens in yeast cells deficient in RAD3, an XPD homolog; such cells exhibit a hyper-recombination phenotype accompanied by a parallel increase in mutation frequency, which is frequently referred to as the Rem (for recombination/mutation) phenotype (Montelone et al. 1988). In contrast, we found that uvh6-1plants had a lower rate of spontaneous homologous recombination. Similar findings were reported for human XPD-deficient cells (Savolainen et al. 2010). It was observed that these cells were deficient not only in NER, but also in transcription-associated recombination. At the same time, DSB-induced recombination was not altered in these cells. Similarly, the authors showed that cells deficient in the Cockayne syndrome B protein that is involved in transcription-coupled repair are impaired in the same manner: they exhibit an up to 20-fold decrease in the spontaneous recombination frequency, but not in HR efficiency induced by DSBs (Savolainen et al. 2010). Also, XPD overexpression in human SK-MG-4 cancer cells treated with cisplatin leads to increased Rad51-related homologous recombination repair and increased sister chromatid exchanges (Aloyz et al. 2002). Our findings for uvh6-1 plants are very similar: the plants seem to be defective in spontaneous RR, which may largely be attributed to transcription-associated recombination, but they are proficient in stress-induced HR. Thus, because of similarities of our findings with findings in studies of animal XPD, we may assume that, similarly to animal XPD, in plants, the deficiency in recombination repair found in uvh6-1 mutant may be related to the deficiency in transcription. Changes in the expression of genes involved in DNA metabolism in uvh6-1 plants Lower levels of KU70 expression in the mutants may reflect a lower capacity of NHEJ repair observed in the DNA repair assay. Similarly, a lower increase in the expression of RAD51 in response to UVC also correlated with the fact that an increase in HRF in response to UVC was also lower in the mutants (Fig. 3). The inefficient NER may result in the accumulation of unrepaired intermediates and an increase in strand breaks. Indeed, we observed the uvh6-1 plants to have much higher levels of strand breaks. Strand breaks are processed by NHEJ and HR repair mechanisms. Our analysis showed that spontaneous PMF was increased, whereas spontaneous HRF was decreased.

Thus, the uvh6-1 plants seem to be also impaired in strand break repair. In animals, during replication, NER-deficient cells can catalyze DNA incisions around bulky lesions, but they fail to perform the post-incision steps retaining TFIIH at the site of damage. The recombination repair rescues broken replication forks via the replication restart mechanisms, one of them being dependent on Rad51 (MorielCarretero and Aguilera 2010). We did not observe higher levels of RAD51 expression or higher levels of HRF. In the light of these observations, it can be hypothesized that the replication restart mechanism is not as efficient in plants as it is in animals. uvh6-1 plants are partially impaired in stress-induced changes in HRF in somatic cells and in the progeny Our previous data indicate that the progeny of plants exposed to salt and heat stress exhibit hypomethylation in the promoter regions of XPD homologs (Boyko et al. 2010a). We hypothesize that in Arabidopsis, changes in methylation and expression of XPD may represent a transgenerational response to stress. A detailed analysis of HRF in the progeny of wild-type and uvh6-1 plants stressed with UVC, cold, heat, flood and drought showed a lower increase in HRF as compared to that in wild-type plants in response to different types of stresses except flood (p \ 0.05 for all types of stresses except flood). Thus, Arabidopsis UVH6, a homolog of human XPD, may indeed play a certain role in transgenerational responses in the form of changes in genome stability, mainly, HRF. We have previously demonstrated that UVC, salt, cold, heat, flood, drought and virus infection lead to a variety of changes in genome stability in the progeny, including an increase in PMR, HRF and microsatellite instability (Boyko and Kovalchuk 2010, 2011a, b; Kathiria et al. 2010; Yao and Kovalchuk 2011). We have also found that the siRNA biogenesis machinery and DNA methylation may be necessary for the establishment of transgenerational changes (Boyko et al. 2010a). A recent report also indicates the importance of siRNA biogenesis for transgenerational changes (Ito et al. 2011). Our current work suggests that UVH6 may play a certain role in transgenerational increase in HRF in response to several abiotic stresses.

Conclusion Our data demonstrate that uvh6-1 may be impaired in both SSB/DSB repair and excision repair. The higher level of strand breaks observed in uvh6-1 plants does not lead to higher HRF. It can be hypothesized that UVH6 may be an essential component of homologous recombination repair.

123

Plant Cell Rep

Finally, we show that uvh6-1 plants are partially deficient in stress-induced transgenerational changes in HRF, which further implies a role of UVH6 in HR repair. Acknowledgments We thank Valentina Titova for proofreading the manuscript. IK acknowledges the financial support of National Science and Engineering Research Council of Canada and Alberta Innovates. We thank Jennifer D. Hall for the gift of uvh6-1 seeds. Conflict of interest of interest.

The authors declare that they have no conflict

References Aloyz R, Xu ZY, Bello V, Bergeron J, Han FY, Yan Y, Malapetsa A, Alaoui-Jamali MA, Duncan AM, Panasci L (2002) Regulation of cisplatin resistance and homologous recombinational repair by the TFIIH subunit XPD. Cancer Res 62:5457–5462 Boyko A, Kovalchuk I (2010) Transgenerational response to stress in Arabidopsis thaliana. Plant Signal Behav 5:995–998 Boyko A, Kovalchuk I (2011a) Genetic and epigenetic effects of plant–pathogen interactions: an evolutionary perspective. Mol Plant 4(6):1014–1023 Boyko A, Kovalchuk I (2011b) Genome instability and epigenetic modification-heritable responses to environmental stress? Curr Opin Plant Biol 14(3):260–266 Boyko A, Greer M, Kovalchuk I (2006a) Acute exposure to UVB has a more profound effect on plant genome stability than chronic exposure. Mutat Res 602:100–109 Boyko A, Hudson D, Bhomkar P, Kathiria P, Kovalchuk I (2006b) Increase of homologous recombination frequency in vascular tissue of Arabidopsis plants exposed to salt stress. Plant Cell Physiol 47:736–742 Boyko A, Zemp F, Filkowski J, Kovalchuk I (2006c) Double-strand break repair in plants is developmentally regulated. Plant Physiol 141:488–497 Boyko A, Blevins T, Yao Y, Golubov A, Bilichak A, Ilnytskyy Y, Hollunder J, Meins F Jr, Kovalchuk I (2010a) Transgenerational adaptation of Arabidopsis to stress requires DNA methylation and the function of Dicer-like proteins. PLoS One 5:e9514 Boyko A, Golubov A, Bilichak A, Kovalchuk I (2010b) Chlorine ions but not sodium ions alter genome stability of Arabidopsis thaliana. Plant Cell Physiol 51:1066–1078 Collins AR (2004) The comet assay for DNA damage and repair: principles, applications, and limitations. Mol Biotechnol 26:249–261 Costa RM, Morgante PG, Berra CM, Nakabashi M, Bruneau D, Bouchez D, Sweder KS, Van Sluys MA, Menck CF (2001) The participation of AtXPB1, the XPB/RAD25 homologue gene from Arabidopsis thaliana, in DNA repair and plant development. Plant J 28:385–395 de Boer J, Hoeijmakers JH (2000) Nucleotide excision repair and human syndromes. Carcinogenesis 21:453–460 Golubov A, Yao Y, Maheshwari P, Bilichak A, Boyko A, Belzile F, Kovalchuk I (2010) Microsatellite instability in Arabidopsis increases with plant development. Plant Physiol 154:1415–1427 Hall JD, Cobb J, Iqbal M, Abidali M, Liu Z, David W (2009) Mount UVH6, a plant homolog of the human/yeast TFIIH transcription factor subunit XPD/RAD3, regulates cold-stress genes in Arabidopsis thaliana. Plant Mol Biol Rep 27:217–228

123

Hays JB (2002) Arabidopsis thaliana, a versatile model system for study of eukaryotic genome-maintenance functions. DNA Repair (Amst) 1:579–600 Ilnytskyy Y, Boyko A, Kovalchuk I (2004) Luciferase-based transgenic recombination assay is more sensitive than beta-glucoronidase-based. Mutat Res 559:189–197 Ilnytskyy Y, Yao Y, Kovalchuk I (2005) Double-strand break repair machinery is sensitive to UV radiation. J Mol Biol 345:707–715 Ito H, Gaubert H, Bucher E, Mirouze M, Vaillant I, Paszkowski J (2011) An siRNA pathway prevents transgenerational retrotransposition in plants subjected to stress. Nature 472:115–119 Jenkins ME, Harlow GR, Liu Z, Shotwell MA, Ma J, Mount DW (1995) Radiation-sensitive mutants of Arabidopsis thaliana. Genetics 140:725–732 Jenkins ME, Suzuki TC, Mount DW (1997) Evidence that heat and ultraviolet radiation activate a common stress-response program in plants that is altered in the uvh6 mutant of Arabidopsis thaliana. Plant Physiol 115:1351–1358 Kathiria P, Sidler C, Golubov A, Kalischuk M, Kawchuk LM, Kovalchuk I (2010) Tobacco mosaic virus infection results in an increase in recombination frequency and resistance to viral, bacterial, and fungal pathogens in the progeny of infected tobacco plants. Plant Physiol 153:1859–1870 Kazimirova A, Barancokova M, Dzupinkova Z, Wsolova L, Dusinska M (2009) Micronuclei and chromosomal aberrations, important markers of ageing: possible association with XPC and XPD polymorphisms. Mutat Res 661:35–40 Kovalchuk I, Kovalchuk O, Hohn B (2000a) Genome-wide variation of the somatic mutation frequency in transgenic plants. EMBO J 19:4431–4438 Kovalchuk O, Arkhipov A, Barylyak I, Karachov I, Titov V, Hohn B, Kovalchuk I (2000b) Plants experiencing chronic internal exposure to ionizing radiation exhibit higher frequency of homologous recombination than acutely irradiated plants. Mutat Res 449:47–56 Kozak J, West CE, White C, da Costa-Nunes JA, Angelis KJ (2009) Rapid repair of DNA double strand breaks in Arabidopsis thaliana is dependent on proteins involved in chromosome structure maintenance. DNA Repair 8:413–419 Kunz BA, Cahill DM, Mohr PG, Osmond MJ, Vonarx EJ (2006) Plant responses to UV radiation and links to pathogen resistance. Int Rev Cytol 255:1–40 Larkindale J, Hall JD, Knight MR, Vierling E (2005) Heat stress phenotypes of Arabidopsis mutants implicate multiple signaling pathways in the acquisition of thermotolerance. Plant Physiol 138:882–897 Lehmann AR (2001) The xeroderma pigmentosum group D (XPD) gene: one gene, two functions, three diseases. Genes Dev 15:15–23 Li A, Schuermann D, Gallego F, Kovalchuk I, Tinland B (2002) Repair of damaged DNA by Arabidopsis cell extract. Plant Cell 14:263–273 Liu Z, Hossain GS, Islas-Osuna MA, Mitchell DL, Mount DW (2000) Repair of UV damage in plants by nucleotide excision repair: arabidopsis UVH1 DNA repair gene is a homolog of Saccharomyces cerevisiae Rad1. Plant J 21:519–528 Liu Z, Hong SW, Escobar M, Vierling E, Mitchell DL, Mount DW, Hall JD (2003) Arabidopsis UVH6, a homolog of human XPD and yeast RAD3 DNA repair genes, functions in DNA repair and is essential for plant growth. Plant Physiol 132:1405–1414 Malone RE, Hoekstra MF (1984) Relationships between a hyper-rec mutation (REM1) and other recombination and repair genes in yeast. Genetics 107:33–48 Montelone BA, Hoekstra MF, Malone RE (1988) Spontaneous mitotic recombination in yeast: the hyper-recombinational

Plant Cell Rep rem1 mutations are alleles of the RAD3 gene. Genetics 119:289–301 Moriel-Carretero M, Aguilera A (2010) A postincision-deficient TFIIH causes replication fork breakage and uncovers alternative Rad51- or Pol32-mediated restart mechanisms. Mol Cell 37:690–701 Oh KS, Bustin M, Mazur SJ, Appella E, Kraemer KH (2011) UVinduced histone H2AX phosphorylation and DNA damage related proteins accumulate and persist in nucleotide excision repair-deficient XP-B cells. DNA Repair (Amst) 10:5–15 Prakash S, Prakash L (2000) Nucleotide excision repair in yeast. Mutat Res 451:13–24 Puchta H (2005) The repair of double-strand breaks in plants: mechanisms and consequences for genome evolution. J Exp Bot 56:1–14 Ries G, Buchholz G, Frohnmeyer H, Hohn B (2000) UV-damagemediated induction of homologous recombination in Arabidopsis is dependent on photosynthetically active radiation. Proc Natl Acad Sci USA 97:13425–13429 Savolainen L, Cassel T, Helleday T (2010) The XPD subunit of TFIIH is required for transcription-associated but not DNA double-strand break-induced recombination in mammalian cells. Mutagenesis 25:623–629 Swoboda P, Hohn B, Gal S (1993) Somatic homologous recombination in planta: the recombination frequency is dependent on the allelic state of recombining sequences and may be influenced by genomic position effects. Mol Gen Genet 237:33–40 Tuteja N, Singh MB, Misra MK, Bhalla PL, Tuteja R (2001) Molecular mechanisms of DNA damage and repair: progress in plants. Crit Rev Biochem Mol Biol 36:337–397

Van der Auwera G, Baute J, Bauwens M, Peck I, Piette D, Pycke M, Asselman P, Depicker A (2008) Development and application of novel constructs to score C:G-to-T: A transitions and homologous recombination in Arabidopsis. Plant Physiol 146:22–31 Vannier JB, Depeiges A, White C, Gallego ME (2009) ERCC1/XPF protects short telomeres from homologous recombination in Arabidopsis thaliana. PLoS Genet 5:e1000380 Vonarx EJ, Tabone EK, Osmond MJ, Anderson HJ, Kunz BA (2006) Arabidopsis homologue of human transcription factor IIH/ nucleotide excision repair factor p44 can function in transcription and DNA repair and interacts with AtXPD. Plant J 46:512–521 Wu FH, Shen SC, Lee LY, Lee SH, Chan MT, Lin CS (2009) TapeArabidopsis Sandwich: a simpler Arabidopsis protoplast isolation method. Plant Methods 5:16 Yao Y, Kovalchuk I (2011) Abiotic stress leads to somatic and heritable changes in homologous recombination frequency, point mutation frequency and microsatellite stability in Arabidopsis plants. Mutat Res 707:61–66 Yao Y, Bilichak A, Golubov A, Blevins T, Kovalchuk I (2010) Differential sensitivity of Arabidopsis siRNA biogenesis mutants to genotoxic stress. Plant Cell Rep 29:1401–1410 Yao Y, Bilichak A, Golubov A, Kovalchuk I (2011) Local infection with oilseed rape mosaic virus promotes genetic rearrangements in systemic Arabidopsis tissue. Mutat Res 709–710:7–14 Yoo SD, Cho YH, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2:1565–1572

123

Genome stability in the uvh6 mutant of Arabidopsis thaliana.

Plant XPD homolog UVH6 is the protein involved in the repair of strand breaks, and the excision repair and uvh6 mutant is not impaired in transgenerat...
785KB Sizes 1 Downloads 3 Views