The AtRAD21.1 and AtRAD21.3 Arabidopsis cohesins play a synergistic role in somatic DNA double strand break damage repair

Background The RAD21 cohesin plays, besides its well-recognised role in chromatid cohesion, a role in DNA double strand break (dsb) repair. In Arabidopsis there are three RAD21 paralog genes (AtRAD21.1, AtRAD21.2 and AtRAD21.3), yet only AtRAD21.1 has been shown to be required for DNA dsb damage repair. Further investigation of the role of cohesins in DNA dsb repair was carried out and is here reported. Results We show for the first time that not only AtRAD21.1 but also AtRAD21.3 play a role in somatic DNA dsb repair. Comet data shows that the lack of either cohesins induces a similar high basal level of DNA dsb in the nuclei and a slower DNA dsb repair kinetics in both cohesin mutants. The observed AtRAD21.3 transcriptional response to DNA dsb induction reinforces further the role of this cohesin in DNA dsb repair. The importance of AtRAD21.3 in DNA dsb damage repair, after exposure to DNA dsb damage inducing agents, is notorious and recognisably evident at the phenotypical level, particularly when the AtRAD21.1 gene is also disrupted. Data on the kinetics of DNA dsb damage repair and DNA damage sensitivity assays, of single and double atrad21 mutants, as well as the transcription dynamics of the AtRAD21 cohesins over a period of 48 hours after the induction of DNA dsb damage is also shown. Conclusions Our data demonstrates that both Arabidopsis cohesin (AtRAD21.1 and AtRAD21.3) play a role in somatic DNA dsb repair. Furthermore, the phenotypical data from the atrad21.1 atrad21.3 double mutant indicates that these two cohesins function synergistically in DNA dsb repair. The implications of this data are discussed. Electronic supplementary material The online version of this article (doi:10.1186/s12870-014-0353-9) contains supplementary material, which is available to authorized users.


qRT-PCR reaction was performed in the iQ™5 Real-Time PCR Detection
System, in a 20l reaction mix containing cDNA corresponding to 6.1ng of total RNA, 250nM of each primer and 10 µl of iQ™ SYBR Green Supermix (Bio-Rad). Quantification of gene expression was carried out after PCR amplification (1 cycle, 95°C for 3 minutes; 45 Cycles, 95°C for 10 seconds, 60°C for 10 seconds, 72°C for 10 seconds (with plate read); 1 Cycle, Melting curve from 55°C to 95.5°C, reading every 0.5°C, hold 10 seconds). Three different qRT-PCR reactions from each of the three cDNA replicas were carried out and quantified. For a given time point after the irradiation, the expression value attributed to each gene is the average from the three independent biological replicas. For each gene, the data was normalised using the expression level of non-irradiated samples as reference (arbitrary value of 1). Actin2 [3] and AtEF1αA4 [4] were used as reference genes. Relative quantification of transcript accumulation of the genes of interest, using Actin2 and AtEF1αA4 as reference genes, was obtained using the Pfaffl method [5]. Reference values for inter-plate calibration were obtained by amplifying, with the Actin2 primers, a dilution series of a bulk mix of all cDNA samples. The quantification cycle (Cq) was determined using Bio-Rad iQ5 Optical System Software, Version 2.0, Standard Edition.
The primers (with a calculated Tm of approximately 60C) were designed with the aid of the Netprimer program. Primers were empirically tested; the melting curve was analysed (iQ™5 Real-Time PCR) and the size and number of the PCR products assessed (not shown) in agarose gels. All the primer pairs promote PCR amplification of single PCR products with efficiency between 90% and 106%. Primer pairs sequence and additional information is provided here in, in Additional file Table S3. Each of the primers qRTmR21-1L, qRTR21-1R, qRTR21-2Rz, qRTR21-3Lx, qRTR21-3Rx, qRTR51-L and rtATM-R spans two adjacent exons. The primer qRTEF1-Lx spans the 5'UTR and the 1 st exon. The two qRT-PCR amplicons from AtRAD21.1 span the 9 th and 10 th exons (87bp), and the 12 th and 13 th exons (110bp). The amplicon from AtRAD21.2 spans the 7 th and 8 th exons (108bp), and the one from AtRAD21.3 spans the 12 th , 13 th and 14 th exons (132bp). The amplicon from AtRAD51 spans the 1 st and 2 nd exons, and that of the AtATM spans the 45 th , 46 th and 47 th exons. Both the qRT-PCR amplicon from Actin2 and that from AtEF1αA4 span the 5'UTRs and the 1 st exon. The regions of the AtRAD21 genes that are amplified (and the respective amplicon length) with the qRT-PCR primer pairs are depicted in Additional file Figure   S1f).

Comet assay
Seeds were germinated under a 16 hours of light (at 22° C) followed by 8 hours of darkness (at 18° C) cycle, on Petri dishes containing ½MS media with Gamborg B5 Vitamins (Duchefa, Haarlem, The Netherlands) solidified with 0.8% Plant agar (Duchefa, Haarlem, The Netherlands) and overlaid with cellophane to facilitate collection of seedlings. Prior treatment, seedlings were gently transferred from cellophane to liquid ½MS media, to avoid dehydration.
Nuclear DNA fragmentation was measured in 10-days-old seedlings untreated and treated with 10 µg/ml or 30 µg/ml Bleomycin Sulfate (Bleomedac; Hamburg, Germany) for 1 hour in liquid ½MS. After Bleomycin treatment, seedlings were thoroughly rinsed in H 2 O, blotted on filter paper and either immediately flash frozen in liquid nitrogen (t = 0) or left to recover in ½MS for the indicated repair times, before being frozen. DNA double strand breaks (dsb) were assayed using a neutral comet assay [6,7]. Plant material processing and data acquisition was carried out as described in previous reports [7]. In brief 70 µl of nuclear suspension obtained by chopping seedlings with a razor blade were dispersed in 280 µl of melted 0.7% LMT agarose (GibcoBRL, Gaithersburg, USA) and used to cast four gels on two microscopic slides per sample analysed.   to ionising radiation ( + ) (a DNA dsb inducing agent). The higher levels of expression of the AtRAD51 gene in the Het and Comp samples confirm that these samples were exposed to ionising radiation ( + ) (d) [1]. Beta Tubulin gene family expression data was used as control, showing that the detected differences in gene expression levels are not due to differences in cDNA template input (e) [2]. Samples non-exposed to DNA dsb inducing agents (Col -) have low/undetectable AtRAD21.1 transcript content [3].

Genotypes:
Col -wild-type Columbia-0 plant Samples Col and Het were harvested from rosette leaves; samples from Comp were harvested from seedlings. According to previous reports [3], both tissues (seedlings and rosette leaves) have undetectable or low transcript levels of alleles (in heterozygous (Het) AtRAD21.1 atrad21.1) in plants exposed and nonexposed to γ-rays. Primer pair atrad51cU and atrad51cD attests which samples were non-exposed (low expression) and exposed (high expression) to DNA dsb ionising radiation (d). TUB-L -TUB-R PCR products were used to certify that the cDNA input was identical in all PCR reactions (e).
Vertical line (in image A) separates two different PCR products photographed in the same gel.  Sequence of the primer pairs amplifying the qRT-PCR amplicons (51, 1, 1m, z, 3 and at) is provided in Additional file Table S3, and qRT-PCR data is provided in Additional file Table S2.

Figure S4
Mean of the number of true leaves per seedling after exposure to ionising radiation Both the median (Figure 4) and the mean (Additional files Figure S4 and The reduced number of true leaves per seedling, that is characteristic to plants exposed to DNA damage inducing ionising radiation, is illustrated in Figure 3 as well as in other sources [1,2]. Error bars represent the standard deviation of the data above and below the mean. Black asterisk denotes significant difference between wild type Columbia-0 (Col) and atrad21.  .3 (112). The total number of seedlings is shown between brackets. 0 Gy -non-exposed to ionising radiation. 100 Gy, 150 Gy -exposed to 100 Gy (or 150 Gy) of ionising radiation (γ-rays; 3.25 Gy/minute; source: Cs137). Col -Wild  The amplicon (AtRAD21.1 allele specific) amplified with the primers 51L and 51R has a higher molecular weight than the amplicon (atrad21.1 allele specific) amplified with the 51L and LBa1 primers. Primers sequence is provided in Additional file Table S7.
Due to the detection of skewed data distributions at 100 Gy and 150 Gy, the values of the Median and Mode are shown to illustrate better the higher incidence of seedlings with 0 true leaves, 15 days after the irradiation, in the genotypes that are more hypersensitive to ionising radiation exposure. The Mean, Median and Mode obtained from the combined data (Additional file   Table S4) from both independent biological replicas (assays) combined. σ upstandard deviation of data with equal or higher value than the mean (or median); σ down -standard deviation of data with lower value than the mean (or median).