NSE5 subunit interacts with distant regions of the SMC arms in the Physcomitrium patens SMC5/6 complex.

Structural maintenance of chromosome (SMC) complexes play roles in cohesion, condensation, replication, transcription, and DNA repair. Their cores are composed of SMC proteins with a unique structure consisting of an ATPase head, long arm, and hinge. SMC complexes form long rod-like structures, which can change to ring-like and elbow-bent conformations upon binding ATP, DNA, and other regulatory factors. These SMC dynamic conformational changes are involved in their loading, translocation, and DNA loop extrusion. Here, we examined the binding and role of the PpNSE5 regulatory factor of Physcomitrium patens PpSMC5/6 complex. We found that the PpNSE5 C-terminal half (aa230-505) is required for binding to its PpNSE6 partner, while the N-terminal half (aa1-230) binds PpSMC subunits. Specifically, the first 71 amino acids of PpNSE5 were required for binding to PpSMC6. Interestingly, the PpNSE5 binding required the PpSMC6 head-proximal joint region and PpSMC5 hinge-proximal arm, suggesting a long distance between binding sites on PpSMC5 and PpSMC6 arms. Therefore, we hypothesize that PpNSE5 either links two antiparallel SMC5/6 complexes or binds one SMC5/6 in elbow-bent conformation, the later model being consistent with the role of NSE5/NSE6 dimer as SMC5/6 loading factor to DNA lesions. In addition, we generated the P. patens Ppnse5KO1 mutant line with an N-terminally truncated version of PpNSE5, which exhibited DNA repair defects while keeping a normal number of rDNA repeats. As the first 71 amino acids of PpNSE5 are required for PpSMC6 binding, our results suggest the role of PpNSE5-PpSMC6 interaction in SMC5/6 loading to DNA lesions.

Characterization of the conserved features of the NSE6 subunit of the Physcomitrium patens SMC5/6 complex.

Structural maintenance of chromosomes (SMC) complexes are molecular machines ensuring chromatin organization at higher levels. They play direct roles in cohesion, condensation, replication, transcription, and DNA repair. Their cores are composed of long-armed SMC, kleisin, and kleisin-associated subunits. Additional factors, like NSE6 within SMC5/6, bind to SMC core complexes and regulate their activities. In the human HsNSE6/SLF2, we recently identified a new CANIN domain. Here we tracked down its sequence homology to lower plants, selected the bryophyte Physcomitrium patens, and analyzed PpNSE6 protein-protein interactions to explore its conservation in detail. We identified a previously unrecognized core sequence motif conserved from yeasts to humans within the NSE6 CANIN domain. This motif mediates the interaction between NSE6 and its NSE5 partner in yeasts and plants. In addition, the CANIN domain and its preceding PpNSE6 sequences bind both PpSMC5 and PpSMC6 arms. Interestingly, we mapped the PpNSE6-binding site at the PpSMC5 arm right next to the PpNSE2-binding surface. The position of NSE6 at SMC arms suggests its role in the regulation of SMC5/6 dynamics. Consistent with the regulatory role of NSE6 subunits, Ppnse6 mutant lines were viable and sensitive to the DNA-damaging drug bleomycin and lost a large portion of rDNA copies. These moss mutants also exhibited reduced growth and developmental aberrations. Altogether, our data showed the conserved function of the NSE6 subunit and architecture of the SMC5/6 complex across species.

RAD51 and RAD51B Play Diverse Roles in the Repair of DNA Double Strand Breaks in Physcomitrium patens.

RAD51 is involved in finding and invading homologous DNA sequences for accurate homologous recombination (HR). Its paralogs have evolved to regulate and promote RAD51 functions. The efficient gene targeting and high HR rates are unique in plants only in the moss Physcomitrium patens (P. patens). In addition to two functionally equivalent RAD51 genes (RAD1-1 and RAD51-2), other RAD51 paralogues were also identified in P. patens. For elucidation of RAD51's involvement during DSB repair, two knockout lines were constructed, one mutated in both RAD51 genes (Pprad51-1-2) and the second with mutated RAD51B gene (Pprad51B). Both lines are equally hypersensitive to bleomycin, in contrast to their very different DSB repair efficiency. Whereas DSB repair in Pprad51-1-2 is even faster than in WT, in Pprad51B, it is slow, particularly during the second phase of repair kinetic. We interpret these results as PpRAD51-1 and -2 being true functional homologs of ancestral RAD51 involved in the homology search during HR. Absence of RAD51 redirects DSB repair to the fast NHEJ pathway and leads to a reduced 5S and 18S rDNA copy number. The exact role of the RAD51B paralog remains unclear, though it is important in damage recognition and orchestrating HR response.

Kleisin NSE4 of the SMC5/6 complex is necessary for DNA double strand break repair, but not for recovery from DNA damage in Physcomitrella (Physcomitrium patens).

KEY MESSAGE: Kleisin NSE4 and circular form of SMC5/6 is indispensable for DSB repair and necessary for gene targeting but is not enough for recovery of cells from DNA damage in Physcomitrella. Structural maintenance of chromosomes (SMC) complexes are involved in cohesion, condensation and maintenance of genome stability. Based on the sensitivity of mutants to genotoxic stress the SMC5/6 complex is thought to play a prominent role in DNA stabilization during repair by tethering DNA at the site of lesion by a heteroduplex of SMC5 and SMC6 encircled with non-SMC components NSE1, NSE3 and kleisin NSE4. In this study, we tested how formation of the SMC5/6 circular structure affects mutant sensitivity to DNA damage, kinetics of DSB repair and gene targeting. In the moss Physcomitrella (Physcomitrium patens), SMC6 and NSE4 are essential single copy genes and this is why we used blocking of transcription to reveal their mutated phenotype. Even slight reduction of transcript levels by dCas9 binding was enough to obtain stable lines with severe DSB repair defects and specific bleomycin sensitivity. We show that survival after bleomycin or MMS treatment fully depends on active SMC6, whereas attenuation of NSE4 has little or negligible effect. We conclude that circularization of SMC5/6 provided by the kleisin NSE4 is indispensable for the DSB repair, nevertheless there are other functions associated with the SMC5/6 complex, which are critical to survive DNA damage.

DNA damage triggers reprogramming of differentiated cells into stem cells in Physcomitrella.

DNA damage can result from intrinsic cellular processes and from exposure to stressful environments. Such DNA damage generally threatens genome integrity and cell viability1. However, here we report that the transient induction of DNA strand breaks (single-strand breaks, double-strand breaks or both) in the moss Physcomitrella patens can trigger the reprogramming of differentiated leaf cells into stem cells without cell death. After intact leafy shoots (gametophores) were exposed to zeocin, an inducer of DNA strand breaks, the STEM CELL-INDUCING FACTOR 1 (STEMIN1)2 promoter was activated in some leaf cells. These cells subsequently initiated tip growth and underwent asymmetric cell divisions to form chloronema apical stem cells, which are in an earlier phase of the life cycle than leaf cells and have the ability to form new gametophores. This DNA-strand-break-induced reprogramming required the DNA damage sensor ATR kinase, but not ATM kinase, together with STEMIN1 and closely related proteins. ATR was also indispensable for the induction of STEMIN1 by DNA strand breaks. Our findings indicate that DNA strand breaks, which are usually considered to pose a severe threat to cells, trigger cellular reprogramming towards stem cells via the activity of ATR and STEMINs.

Roles of RAD51 and RTEL1 in telomere and rDNA stability in Physcomitrella patens.

Telomeres and ribosomal RNA genes (rDNA) are essential for cell survival and particularly sensitive to factors affecting genome stability. Here, we examine the role of RAD51 and its antagonist, RTEL1, in the moss Physcomitrella patens. In corresponding mutants, we analyse their sensitivity to DNA damage, the maintenance of telomeres and rDNA, and repair of double-stranded breaks (DSBs) induced by genotoxins with various modes of action. While the loss of RTEL1 results in rapid telomere shortening, concurrent loss of both RAD51 genes has no effect on telomere lengths. We further demonstrate here the linked arrangement of 5S and 45S rRNA genes in P. patens. The spacer between 5S and 18S rRNA genes, especially the region downstream from the transcription start site, shows conspicuous clustering of sites with a high propensity to form quadruplex (G4) structures. Copy numbers of 5S and 18S rDNA are reduced moderately in the pprtel1 mutant, and significantly in the double pprad51-1-2 mutant, with no progression during subsequent cultivation. While reductions in 45S rDNA copy numbers observed in pprtel1 and pprad51-1-2 plants apply also to 5S rDNA, changes in transcript levels are different for 45S and 5S rRNA, indicating their independent transcription by RNA polymerase I and III, respectively. The loss of SOL (Sog One-Like), a transcription factor regulating numerous genes involved in DSB repair, increases the rate of DSB repair in dividing as well as differentiated tissue, and through deactivation of G2/M cell-cycle checkpoint allows the cell-cycle progression manifested as a phenotype resistant to bleomycin.

Integrating plant and animal biology for the search of novel DNA damage biomarkers.

Eukaryotic genome surveillance is dependent on the multiple, highly coordinated network functions of the DNA damage response (DDR). Highlighted conserved features of DDR in plants and animals represent a challenging opportunity to develop novel interdisciplinary investigations aimed at expanding the sets of DNA damage biomarkers currently available for radiation exposure monitoring (REM) in environmental and biomedical applications. In this review, common and divergent features of the most relevant DDR players in animals and plants are described, including the intriguing example of the plant and animal kingdom-specific master regulators SOG1 (suppressor of gamma response) and p53. The potential of chromatin remodelers as novel predictive biomarkers of DNA damage is considered since these highly evolutionarily conserved proteins provide a docking platform for the DNA repair machinery. The constraints of conventional REM biomarkers can be overcome using biomarkers identified with the help of the pool provided by high-throughput techniques. The complexity of radiation-responsive animal and plant transcriptomes and their usefulness as sources of novel REM biomarkers are discussed, focusing on ionizing (IR) and UV-radiation. The possible advantages resulting from the exploitation of plants as sources of novel DNA damage biomarkers for monitoring the response to radiation-mediated genotoxic stress are listed. Plants could represent an ideal system for the functional characterization of knockout mutations in DDR genes which compromise cell survival in animals. However, the pronounced differences between plant and animal cells need to be carefully considered in order to avoid any misleading interpretations. Radioresistant plant-based systems might be useful to explore the molecular bases of LD (low dose)/LDR (low dose rate) responses since nowadays it is extremely difficult to perform an accurate assessment of LD/LDR risk to human health. To overcome these constraints, researchers have started exploring radiotolerant non-human species as potential sources of information on the mechanisms involved in LD/LDR and general radiation responses.

Evolutionarily Distant Streptophyta Respond Differently to Genotoxic Stress.

Research in algae usually focuses on the description and characterization of morpho-and phenotype as a result of adaptation to a particular habitat and its conditions. To better understand the evolution of lineages we characterized responses of filamentous streptophyte green algae of the genera Klebsormidium and Zygnema, and of a land plant-the moss Physcomitrellapatens-to genotoxic stress that might be relevant to their environment. We studied the induction and repair of DNA double strand breaks (DSBs) elicited by the radiomimetic drug bleomycin, DNA single strand breaks (SSB) as consequence of base modification by the alkylation agent methyl methanesulfonate (MMS) and of ultra violet (UV)-induced photo-dimers, because the mode of action of these three genotoxic agents is well understood. We show that the Klebsormidium and Physcomitrella are similarly sensitive to introduced DNA lesions and have similar rates of DSBs repair. In contrast, less DNA damage and higher repair rate of DSBs was detected in Zygnema, suggesting different mechanisms of maintaining genome integrity in response to genotoxic stress. Nevertheless, contrary to fewer detected lesions is Zygnema more sensitive to genotoxic treatment than Klebsormidium and Physcomitrella.

Bridging Plant and Human Radiation Response and DNA Repair through an In Silico Approach.

The mechanisms of response to radiation exposure are conserved in plants and animals. The DNA damage response (DDR) pathways are the predominant molecular pathways activated upon exposure to radiation, both in plants and animals. The conserved features of DDR in plants and animals might facilitate interdisciplinary studies that cross traditional boundaries between animal and plant biology in order to expand the collection of biomarkers currently used for radiation exposure monitoring (REM) in environmental and biomedical settings. Genes implicated in trans-kingdom conserved DDR networks often triggered by ionizing radiation (IR) and UV light are deposited into biological databases. In this study, we have applied an innovative approach utilizing data pertinent to plant and human genes from publicly available databases towards the design of a 'plant radiation biodosimeter', that is, a plant and DDR gene-based platform that could serve as a REM reliable biomarker for assessing environmental radiation exposure and associated risk. From our analysis, in addition to REM biomarkers, a significant number of genes, both in human and Arabidopsis thaliana, not yet characterized as DDR, are suggested as possible DNA repair players. Last but not least, we provide an example on the applicability of an Arabidopsis thaliana-based plant system monitoring the role of cancer-related DNA repair genes BRCA1, BARD1 and PARP1 in processing DNA lesions.

Two New Faces of Amifostine: Protector from DNA Damage in Normal Cells and Inhibitor of DNA Repair in Cancer Cells.

Amifostine protects normal cells from DNA damage induction by ionizing radiation or chemotherapeutics, whereas cancer cells typically remain uninfluenced. While confirming this phenomenon, we have revealed by comet assay and currently the most sensitive method of DNA double strand break (DSB) quantification (based on γH2AX/53BP1 high-resolution immunofluorescence microscopy) that amifostine treatment supports DSB repair in γ-irradiated normal NHDF fibroblasts but alters it in MCF7 carcinoma cells. These effects follow from the significantly lower activity of alkaline phosphatase measured in MCF7 cells and their supernatants as compared with NHDF fibroblasts. Liquid chromatography-mass spectrometry confirmed that the amifostine conversion to WR-1065 was significantly more intensive in normal NHDF cells than in tumor MCF cells. In conclusion, due to common differences between normal and cancer cells in their abilities to convert amifostine to its active metabolite WR-1065, amifostine may not only protect in multiple ways normal cells from radiation-induced DNA damage but also make cancer cells suffer from DSB repair alteration.

Arabidopsis DNA polymerase lambda mutant is mildly sensitive to DNA double strand breaks but defective in integration of a transgene.

The DNA double-strand break (DSB) is a critical type of damage, and can be induced by both endogenous sources (e.g., errors of oxidative metabolism, transposable elements, programmed meiotic breaks, or perturbation of the DNA replication fork) and exogenous sources (e.g., ionizing radiation or radiomimetic chemicals). Although higher plants, like mammals, are thought to preferentially repair DSBs via nonhomologous end joining (NHEJ), much remains unclear about plant DSB repair pathways. Our reverse genetic approach suggests that DNA polymerase λ is involved in DSB repair in Arabidopsis. The Arabidopsis T-DNA insertion mutant (atpolλ-1) displayed sensitivity to both gamma-irradiation and treatment with radiomimetic reagents, but not to other DNA damaging treatments. The atpolλ-1 mutant showed a moderate sensitivity to DSBs, while Arabidopsis Ku70 and DNA ligase 4 mutants (atku70-3 and atlig4-2), both of which play critical roles in NHEJ, exhibited a hypersensitivity to these treatments. The atpolλ-1/atlig4-2 double mutant exhibited a higher sensitivity to DSBs than each single mutant, but the atku70/atpolλ-1 showed similar sensitivity to the atku70-3 mutant. We showed that transcription of the DNA ligase 1, DNA ligase 6, and Wee1 genes was quickly induced by BLM in several NHEJ deficient mutants in contrast to wild-type. Finally, the T-DNA transformation efficiency dropped in NHEJ deficient mutants and the lowest transformation efficiency was scored in the atpolλ-1/atlig4-2 double mutant. These results imply that AtPolλ is involved in both DSB repair and DNA damage response pathway.

Telomere dynamics in the lower plant Physcomitrella patens.

A comparative approach in biology is needed to assess the universality of rules governing this discipline. In plant telomere research, most of the key principles were established based on studies in only single model plant, Arabidopsis thaliana. These principles include the absence of telomere shortening during plant development and the corresponding activity of telomerase in dividing (meristem) plant cells. Here we examine these principles in Physcomitrella patens as a representative of lower plants. To follow telomerase expression, we first characterize the gene coding for the telomerase reverse transcriptase subunit PpTERT in P. patens, for which only incomplete prediction has been available so far. In protonema cultures of P. patens, growing by filament apical cell division, the proportion of apical (dividing) cells was quantified and telomere length, telomerase expression and activity were determined. Our results show telomere stability and demonstrate proportionality of telomerase activity and expression with the number of apical cells. In addition, we analyze telomere maintenance in mre11, rad50, nbs1, ku70 and lig4 mutants of P. patens and compare the impact of these mutations in double-strand-break (DSB) repair pathways with earlier observations in corresponding A. thaliana mutants. Telomere phenotypes are absent and DSB repair kinetics is not affected in P. patens mutants for DSB factors involved in non-homologous end joining (NHEJ). This is compliant with the overall dominance of homologous recombination over NHEJ pathways in the moss, contrary to the inverse situation in flowering plants.

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. 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.

Genotoxin induced mutagenesis in the model plant Physcomitrella patens.

The moss Physcomitrella patens is unique for the high frequency of homologous recombination, haploid state, and filamentous growth during early stages of the vegetative growth, which makes it an excellent model plant to study DNA damage responses. We used single cell gel electrophoresis (comet) assay to determine kinetics of response to Bleomycin induced DNA oxidative damage and single and double strand breaks in wild type and mutant lig4 Physcomitrella lines. Moreover, APT gene when inactivated by induced mutations was used as selectable marker to ascertain mutational background at nucleotide level by sequencing of the APT locus. We show that extensive repair of DSBs occurs also in the absence of the functional LIG4, whereas repair of SSBs is seriously compromised. From analysis of induced mutations we conclude that their accumulation rather than remaining lesions in DNA and blocking progression through cell cycle is incompatible with normal plant growth and development and leads to sensitive phenotype.

MRE11 and RAD50, but not NBS1, are essential for gene targeting in the moss Physcomitrella patens.

The moss Physcomitrella patens is unique among plant models for the high frequency with which targeted transgene insertion occurs via homologous recombination. Transgene integration is believed to utilize existing machinery for the detection and repair of DNA double-strand breaks (DSBs). We undertook targeted knockout of the Physcomitrella genes encoding components of the principal sensor of DNA DSBs, the MRN complex. Loss of function of PpMRE11 or PpRAD50 strongly and specifically inhibited gene targeting, whilst rates of untargeted transgene integration were relatively unaffected. In contrast, disruption of the PpNBS1 gene retained the wild-type capacity to integrate transforming DNA efficiently at homologous loci. Analysis of the kinetics of DNA-DSB repair in wild-type and mutant plants by single-nucleus agarose gel electrophoresis revealed that bleomycin-induced fragmentation of genomic DNA was repaired at approximately equal rates in each genotype, although both the Ppmre11 and Pprad50 mutants exhibited severely restricted growth and development and enhanced sensitivity to UV-B and bleomycin-induced DNA damage, compared with wild-type and Ppnbs1 plants. This implies that while extensive DNA repair can occur in the absence of a functional MRN complex; this is unsupervised in nature and results in the accumulation of deleterious mutations incompatible with normal growth and development.

GMI1, a structural-maintenance-of-chromosomes-hinge domain-containing protein, is involved in somatic homologous recombination in Arabidopsis.

DNA double-strand breaks (DSBs) pose one of the most severe threats to genome integrity, potentially leading to cell death. After detection of a DSB, the DNA damage and repair response is initiated and the DSB is repaired by non-homologous end joining and/or homologous recombination. Many components of these processes are still unknown in Arabidopsis thaliana. In this work, we characterized γ-irradiation and mitomycin C induced 1 (GMI1), a member of the SMC-hinge domain-containing protein family. RT-PCR analysis and promoter-GUS fusion studies showed that γ-irradiation, the radio-mimetic drug bleocin, and the DNA cross-linking agent mitomycin C strongly enhance GMI1 expression particularly in meristematic tissues. The induction of GMI1 by γ-irradiation depends on the signalling kinase Ataxia telangiectasia-mutated (ATM) but not on ATM and Rad3-related (ATR). Epistasis analysis of single and double mutants demonstrated that ATM acts upstream of GMI1 while the atr gmi1-2 double mutant was more sensitive than the respective single mutants. Comet assay revealed a reduced rate of DNA double-strand break repair in gmi1 mutants during the early recovery phase after exposure to bleocin. Moreover, the rate of homologous recombination of a reporter construct was strongly reduced in gmi1 mutant plants upon exposure to bleocin or mitomycin C. GMI1 is the first member of its protein family known to be involved in DNA repair.

DNA ligase 1 deficient plants display severe growth defects and delayed repair of both DNA single and double strand breaks.

BACKGROUND: DNA ligase enzymes catalyse the joining of adjacent polynucleotides and as such play important roles in DNA replication and repair pathways. Eukaryotes possess multiple DNA ligases with distinct roles in DNA metabolism, with clear differences in the functions of DNA ligase orthologues between animals, yeast and plants. DNA ligase 1, present in all eukaryotes, plays critical roles in both DNA repair and replication and is indispensable for cell viability. RESULTS: Knockout mutants of atlig1 are lethal. Therefore, RNAi lines with reduced levels of AtLIG1 were generated to allow the roles and importance of Arabidopsis DNA ligase 1 in DNA metabolism to be elucidated. Viable plants were fertile but displayed a severely stunted and stressed growth phenotype. Cell size was reduced in the silenced lines, whilst flow cytometry analysis revealed an increase of cells in S-phase in atlig1-RNAi lines relative to wild type plants. Comet assay analysis of isolated nuclei showed atlig1-RNAi lines displayed slower repair of single strand breaks (SSBs) and also double strand breaks (DSBs), implicating AtLIG1 in repair of both these lesions. CONCLUSION: Reduced levels of Arabidopsis DNA ligase 1 in the silenced lines are sufficient to support plant development but result in retarded growth and reduced cell size, which may reflect roles for AtLIG1 in both replication and repair. The finding that DNA ligase 1 plays an important role in DSB repair in addition to its known function in SSB repair, demonstrates the existence of a previously uncharacterised novel pathway, independent of the conserved NHEJ. These results indicate that DNA ligase 1 functions in both DNA replication and in repair of both ss and dsDNA strand breaks in higher plants.

Rapid repair of DNA double strand breaks in Arabidopsis thaliana is dependent on proteins involved in chromosome structure maintenance.

DNA double strand breaks (DSBs) are one of the most cytotoxic forms of DNA damage and must be repaired by recombination, predominantly via non-homologous joining of DNA ends (NHEJ) in higher eukaryotes. However, analysis of DSB repair kinetics of plant NHEJ mutants atlig4-4 and atku80 with the neutral comet assay shows that alternative DSB repair pathways are active. Surprisingly, these kinetic measurements show that DSB repair was faster in the NHEJ mutant lines than in wild-type Arabidopsis. Here we provide the first characterization of this KU-independent, rapid DSB repair pathway operating in Arabidopsis. The alternate pathway that rapidly removes the majority of DSBs present in nuclear DNA depends upon structural maintenance of chromosomes (SMC) complex proteins, namely MIM/AtRAD18 and AtRAD21.1. An absolute requirement for SMC proteins and kleisin for rapid repair of DSBs in Arabidopsis opens new insight into the mechanism of DSB removal in plants.

The Rad17 homologue of Arabidopsis is involved in the regulation of DNA damage repair and homologous recombination.

Rad17 is involved in DNA checkpoint control in yeast and human cells. A homologue of this gene as well as other genes of the pathway (the 9-1-1 complex) are present in Arabidopsis and share conserved sequence domains with their yeast and human counterparts. DNA-damaging agents induce AtRAD17 transcriptionally. AtRAD17 mutants show increased sensitivity to the DNA-damaging chemicals bleomycin and mitomycin C (MMC), which can be reversed by complementation, suggesting that the loss of function of Rad17 disturbs DNA repair in plant cells. Our results are further confirmed by the phenotype of a mutant of the 9-1-1 complex (Rad9), which is also sensitive to the same chemicals. AtRAD9 and AtRAD17 seem to be epistatic as the double mutant is not more sensitive to the chemicals than the single mutants. The mutants show a delay in the general repair of double-strand breaks (DSBs). However, frequencies of intrachromosomal homologous recombination (HR) are enhanced. Nevertheless, the mutants are proficient for a further induction of HR by genotoxic stresses. Our results indicate that a mutant Rad17 pathway is associated with a general deregulation of DNA repair, which seems to be correlated with a deficiency in non-homologous DSB repair.

BRU1, a novel link between responses to DNA damage and epigenetic gene silencing in Arabidopsis.

DNA repair associated with DNA replication is important for the conservation of genomic sequence information, whereas reconstitution of chromatin after replication sustains epigenetic information. We have isolated and characterized mutations in the BRU1 gene of Arabidopsis that suggest a novel link between these underlying maintenance mechanisms. Bru1 plants are highly sensitive to genotoxic stress and show stochastic release of transcriptional gene silencing. They also show increased intrachromosomal homologous recombination and constitutively activated expression of poly (ADP-ribose) polymerase-2 (AtPARP-2), the induction of which is associated with elevated DNA damage. Bru1 mutations affect the stability of heterochromatin organization but do not interfere with genome-wide DNA methylation. BRU1 encodes a novel nuclear protein with two predicted protein-protein interaction domains. The developmental abnormalities characteristic of bru1 mutant plants resemble those triggered by mutations in genes encoding subunits of chromatin assembly factor (CAF-1), the condensin complex, or MRE11. Comparison of bru1 with these mutants indicates cooperative roles in the replication and stabilization of chromatin structure, providing a novel link between chromatin replication, epigenetic inheritance, S-phase DNA damage checkpoints, and the regulation of meristem development.