Telomere-length regulation in inter-ecotype crosses of Arabidopsis.

Telomeres, the nucleoprotein complexes at the ends of eukaryotic chromosomes, are maintained at a species-specific equilibrium length. Arabidopsis thaliana is a self-fertilizing plant and different geographical isolates or ecotypes show differing telomere-lengths. We have exploited this telomere-length polymorphism between Arabidopsis ecotypes to investigate the genetic regulation of telomere length by analysing telomere lengths in 16 different inter-ecotype crosses between plants with differing telomere sizes. With two exceptions, the inter-ecotype hybrid plants present a new telomere-length set point, intermediate between that of the two parents. A regulation mechanism thus shortens the longer and lengthens the shorter telomeres.

Ku80 plays a role in non-homologous recombination but is not required for T-DNA integration in Arabidopsis.

Chromosomal breaks are repaired by homologous recombination (HR) or non-homologous end joining (NHEJ) mechanisms. The Ku70/Ku80 heterodimer binds DNA ends and plays roles in NHEJ and telomere maintenance in organisms ranging from yeast to humans. We have previously identified a ku80 mutant of the model plant Arabidopsis thaliana and shown the role of Ku80 in telomere homeostasis in plant cells. We show here that this mutant is hypersensitive to the DNA-damaging agent methyl methane sulphonate and has a reduced capacity to carry out NHEJ recombination. To understand the interplay between HR and NHEJ in plants, we measured HR in the absence of Ku80. We find that the frequency of intrachromosomal HR is not affected by the absence of Ku80. Previous work has clearly implicated the Ku heterodimer in Agrobacterium-mediated T-DNA transformation of yeast. Surprisingly, ku80 mutant plants show no defect in the efficiency of T-DNA transformation of plants with Agrobacterium, showing that an alternative pathway must exist in plants.

The plant Rad50-Mre11 protein complex.

The Rad50-Mre11-Xrs2/Nbs1 protein complex plays critical roles in cellular processes involving DNA ends. This complex is implicated in DNA recombination and replication, meiosis, telomere maintenance and cellular DNA damage responses. The Rad50 and Mre11 proteins are essential for viability in animals, although not in yeast. We have prepared antibodies to the Rad50 protein of the model plant Arabidopsis thaliana which recognize a 175 kDa protein in wild-type Arabidopsis protein extracts. Furthermore, we report here demonstration of the existence of the Rad50-Mre11 complex by co-immunoprecipitation of the Rad50 and Mre11 proteins from the plant cell extracts.

Homologous recombination in planta is stimulated in the absence of Rad50.

Chromosomal double-strand DNA breaks must be repaired; in the absence of repair the resulting acentromeric (and telomereless) fragments may be lost and/or the broken DNA ends may recombine causing general chromosomal instability. The Rad50/Mre11/Xrs2 protein complex acts at DNA ends and is implicated in both homologous and non-homologous recombination. We have isolated a rad50 mutant of the plant Arabidopsis thaliana and show here that it has a somatic hyper-recombination phenotype in planta. This finding supports the hypothesis of a competition between homologous and illegitimate recombination in higher eukaryotes. To our knowledge, this is the first direct in vivo support for the role of this complex in chromosomal recombination in a multicellular organism and the first description of a mutation of a known gene leading to hyper-recombination in plants.

Disruption of the Arabidopsis RAD50 gene leads to plant sterility and MMS sensitivity.

The Rad50 protein is involved in the cellular response to DNA-double strand breaks (DSBs), including the detection of damage, activation of cell-cycle checkpoints, and DSB repair via recombination. It is essential for meiosis in yeast, is involved in telomere maintenance, and is essential for cellular viability in mice. Here we present the isolation, sequence and characterization of the Arabidopsis thaliana RAD50 homologue (AtRAD50) and an Arabidopsis mutant of this gene. A single copy of this gene is present in the Arabidopsis genome, located on chromosome II. Northern analysis shows a single 4.3 Kb mRNA species in all plant tissues tested, which is strongly enriched in flowers and other tissues with many dividing cells. The predicted protein presents strong conservation with the other known Rad50 homologues of the amino- and carboxy-terminal regions. Mutant plants present a sterility phenotype which co-segregates with the T-DNA insertion. Molecular analysis of the mutant plants shows that the sterility phenotype is present only in the plants homozygous for the T-DNA insertion. An in vitro mutant cell line, derived from the mutant plant, shows a clear hypersensitivity to the DNA-damaging agent methylmethane sulphonate, suggesting a role of RAD50 in double-strand break repair in plant cells. This is the first report of a plant mutated in a protein of the Rad50-Mre11-Xrs2 complex, as well as the first data suggesting the involvement of the Rad50 homologue protein in meiosis and DNA repair in plants.

RAD50 function is essential for telomere maintenance in Arabidopsis.

We have identified and characterized an Arabidopsis thaliana rad50 mutant plant containing a T-DNA insertion in the AtRAD50 gene and showing both meiotic and DNA repair defects. We report here that rad50/rad50 mutant cells show a progressive shortening of telomeric DNA relative to heterozygous rad50/RAD50 controls and that the mutant cell population rapidly enters a crisis, with the majority of the cells dying. Surviving rad50 mutant cells have longer telomeres than wild-type cells, indicating the existence in plants of an alternative RAD50-independent mechanism for telomere maintenance. These results report the role of a protein essential for double-strand break repair in telomere maintenance in higher eukaryotes.

Positive-negative selection and T-DNA stability in Arabidopsis transformation.

We have analysed the application of positive-negative selection for the selection of homologous recombination interactions between the chromosome and a T-DNA molecule after transformation of plant cells. Two different genomic loci in a cell suspension of Arabidopsis thaliana were chosen to study gene targeting events. One was the chalcone synthase (CHS) gene present as a single copy and the second an hemizygous chromosomally inserted T-DNA containing the hpt gene, conferring resistance to hygromycin, flanked by CHS sequences. The target lines were transformed with replacement-type T-DNA vectors which contained a positive selectable marker flanked by the regions of the CHS gene and a negative selectable marker to counter-select random insertions. As negative marker we used the Escherichia coli codA gene encoding cytosine deaminase, conferring upon the cells sensitivity to 5-flourocytosine (5-FC). Doubly selected transformants represent 1-4% of the primary transformed cells. Targeting events were not found at the chalcone synthase locus nor at the artificial hpt locus in a total of 4379 doubly selected calli, corresponding to at least 109,475 individual primary transformants. We show by PCR and Southern analysis that the 5-FC resistance in the majority of these cells is associated with substantial deletions of the T-DNA molecule from the right-border end.

Mutations in XRS2 and RAD50 delay but do not prevent mating-type switching in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, a large number of genes in the RAD52 epistasis group has been implicated in the repair of chromosomal double-strand breaks and in both mitotic and meiotic homologous recombination. While most of these genes are essential for yeast mating-type (MAT) gene switching, neither RAD50 nor XRS2 is required to complete this specialized mitotic gene conversion process. Using a galactose-inducible HO endonuclease gene to initiate MAT switching, we have examined the effect of null mutations of RAD50 and of XRS2 on intermediate steps of this recombination event. Both rad50 and xrs2 mutants exhibit a marked delay in the completion of switching. Both mutations reduce the extent of 5'-to-3' degradation from the end of the HO-created double-strand break. The steps of initial strand invasion and new DNA synthesis are delayed by approximately 30 min in mutant cells. However, later events are still further delayed, suggesting that XRS2 and RAD50 affect more than one step in the process. In the rad50 xrs2 double mutant, the completion of MAT switching is delayed more than in either single mutant, without reducing the overall efficiency of the process. The XRS2 gene encodes an 854-amino-acid protein with no obvious similarity to the Rad50 protein or to any other protein in the database. Overexpression of RAD50 does not complement the defects in xrs2 or vice versa.

Rapid kinetics of mismatch repair of heteroduplex DNA that is formed during recombination in yeast.

Homothallic switching of yeast mating type (MAT) genes is a highly efficient gene conversion process initiated by a double-strand break. The use of a galactose-inducible HO endonuclease gene has made it possible to analyze the synchronous progression of molecular intermediates during recombination. When MATa switches to MAT alpha, a 3' single-stranded end of HO-cleaved MAT DNA invades the homologous donor, HML alpha, and initiates copying of new DNA sequences. These early steps of recombination can be detected by PCR amplification. When recombination is initiated in a strain carrying the MATa-stk T-->A base pair substitution mutation located 8 bp to the right of the HO endonuclease cleavage site, the stk mutation is frequently included in heteroduplex DNA formed between MAT and HML and undergoes mismatch correction. We have followed the kinetics of mismatch repair of the stk mutation by determining the DNA sequence of the PCR-amplified early intermediates of recombination. Mismatch correction of heteroduplex DNA is quite rapid (t1/2 = 6-10 min) compared to the 60 min required to complete repair of the double-strand break. Mismatch repair occurs soon after the 3'-ended MAT-stk strand invades HML and forms heteroduplex DNA. Moreover, nearly all the correction events are restorations, in which the invading MAT-stk strand is corrected to the genotype of the resident HML donor. This rapid restoration ensures that the net result will be a gene conversion at the MAT locus. Rapid and preferential mismatch repair of heteroduplex DNA has important implications in understanding meiotic recombination.

Heteroduplex formation and mismatch repair of the "stuck" mutation during mating-type switching in Saccharomyces cerevisiae.

We sequenced two alleles of the MATa locus of Saccharomyces cerevisiae that reduce homothallic switching and confer viability to HO rad52 strains. Both the MATa-stk (J. E. Haber, W. T. Savage, S. M. Raposa, B. Weiffenbach, and L. B. Rowe, Proc. Natl. Acad. Sci. USA 77:2824-2828, 1980) and MATa-survivor (R. E. Malone and D. Hyman, Curr. Genet. 7:439-447, 1983) alleles result from a T----A base change at position Z11 of the MAT locus. These strains also contain identical base substitutions at HMRa, so that the mutation is reintroduced when MAT alpha switches to MATa. Mating-type switching in a MATa-stk strain relative to a MATa Z11T strain is reduced at least 50-fold but can be increased by expression of HO from a galactose-inducible promoter. We confirmed by Southern analysis that the Z11A mutation reduced the efficiency of double-strand break formation compared with the Z11T variant; the reduction was more severe in MAT alpha than in MATa. In MAT alpha, the Z11A mutation also creates a mat alpha 1 (sterile) mutation that distinguishes switches of MATa-stk to either MAT alpha or mat alpha 1-stk. Pedigree analysis of cells induced to switch in G1 showed that MATa-stk switched frequently (23% of the time) to produce one mat alpha 1-stk and one MAT alpha progeny. This postswitching segregation suggests that Z11 was often present in heteroduplex DNA that was not mismatch repaired. When mismatch repair was prevented by deletion of the PMS1 gene, there was an increase in the proportion of mat alpha 1-stk/MAT alpha sectors (59%) and in pairs of switched cells that both retained the stk mutation (27%). We conclude that at least one strand of DNA only 4 bp from the HO cut site is not degraded in most of the gene conversion events that accompany MAT switching.

The TSM1 gene of Saccharomyces cerevisiae overlaps the MAT locus.

We have cloned the region from MAT to THR4 on chromosome III of Saccharomyces cerevisiae. Although the region is only 15 kb, the two loci are genetically separated by 22 cM. This is in sharp contrast to the very low level of recombination (2 cM in 22 kb) that is observed in the adjacent CRY1-MAT interval, and suggests that there may be a "hot spot" for recombination in the MAT-THR4 region. The DNA sequence of the first 4.4 kb distal to MAT reveals an open reading frame that we have identified as the essential gene, TSM1. Surprisingly, the TSM1 open reading frame of 1,410 amino acids extends into the MAT locus, such that the 3'-end of the MAT alpha 1 transcript ends 15 bp from the 3'-end of the TSM1 open reading frame.

Intermediates of recombination during mating type switching in Saccharomyces cerevisiae.

We have identified two novel intermediates of homothallic switching of the yeast mating type gene, from MATa to MAT alpha. Following HO endonuclease cleavage, 5' to 3' exonucleolytic digestion is observed distal to the HO cut, creating a 3'-ended single-stranded tail. This recision is more extensive in a rad52 strain unable to switch. Surprisingly, the proximal side of the HO cut is protected from degradation; this stabilization depends on the presence of the silent copy donor sequences. A second intermediate was identified by a quantitative application of the polymerase chain reaction (PCR). The Y alpha-MAT distal covalent fragment of the switched product appears 30 min prior to the appearance of the MAT proximal Y alpha junction. No covalent joining of MAT distal to HML distal sequences is detected. We suggested that the MAT DNA distal to the HO cut invades the intact donor and is extended by DNA synthesis. This step is prevented in a rad52 strain. These intermediates are consistent with a model for MAT switching in which only the distal side of the HO cut is initially active in strand invasion and transfer of information from the donor.

Physical monitoring of mating type switching in Saccharomyces cerevisiae.

The kinetics of mating type switching in Saccharomyces cerevisiae can be followed at the DNA level by using a galactose-inducible HO (GAL-HO) gene to initiate the event in synchronously growing cells. From the time that HO endonuclease cleaves MAT a until the detection of MAT alpha DNA took 60 min. When unbudded G1-phase cells were induced, switched to the opposite mating type in "pairs." In the presence of the DNA synthesis inhibitor hydroxyurea, HO-induced cleavage occurred but cells failed to complete switching. In these blocked cells, the HO-cut ends of MATa remained stable for at least 3 h. Upon removal of hydroxyurea, the cells completed the switch in approximately 1 h. The same kinetics of MAT switching were also seen in asynchronous cultures and when synchronously growing cells were induced at different times of the cell cycle. Thus, the only restriction that confined normal homothallic switching to the G1 phase of the cell cycle was the expression of HO endonuclease. Further evidence that galactose-induced cells can switch in the G2 phase of the cell cycle was the observation that these cells did not always switch in pairs. This suggests that two chromatids, both cleaved with HO endonuclease, can interact independently with the donors HML alpha and HMRa.

Repair of UV-irradiated plasmid DNA in Saccharomyces cerevisiae. Inability to complement mutational defects in excision repair by in vitro treatment with Micrococcus luteus UV endonuclease.

Excision repair defects of Saccharomyces cerevisiae rad1-1, rad4-4, rad7-1 and rad14 mutants were examined. As previously found, transformation of such cells with UV-irradiated plasmid DNA is poor compared to wild-type yeast. Treatment of UV-irradiated YRp12 plasmid DNA with crude preparations of Micrococcus luteus UV endonuclease before introducing it into rad1-1 cells increased transformation efficiency to wild-type levels. This is consistent with earlier reports of rad1-1 mutants being defective in the incision step of excision repair. However, with purified UV endonuclease little or no rescue occurred when the UV-irradiated plasmid was incised before transformation into rad1-1 or rad4-4 cells. Furthermore, the purified UV endonuclease reduced transformation of rad7-1 and rad14 mutants to levels seen in rad1-1 and rad4-4 cells. In contrast such treatment caused only a small decrease in the transforming ability of UV-irradiated DNA in wild-type cells. These results show that yeast can normally process pre-incised, UV-irradiated DNA and that this activity is absent in rad1-1, rad4-4, rad7-1 and rad14 mutants. Thus, in addition to their previously reported roles in incision, the RAD1, 4, 7 and 14 gene products are also required for repair to continue after the incision of DNA lesions.

Induced cellular resistance to ultraviolet light in Saccharomyces cerevisiae is not accompanied by increased repair of plasmid DNA.

Many reports show that resistance of Saccharomyces cerevisiae to a large UV dose can be enhanced by pre-induction with a smaller one given some hours before. This work tests if such increased cell survival is associated with increased DNA repair on UV damaged plasmid transformed into yeast. There was no change in transformation efficiency of UV-damaged plasmid DNA under conditions where RAD cell survival increased 5-fold, and where rad1-1 and rad6-1 survival increased 2-fold. It is concluded that DNA repair activity involving the RAD6 and RAD3 pathways is either not inducible or is unable to work on plasmid DNA. It is suggested that the enhancement of cellular survival detected may be based on changes in cell-cycle behaviour which permit cells generally proficient in repair a greater chance to recover.

The use of plasmid DNA to probe DNA repair functions in the yeast Saccharomyces cerevisiae.

The survival of plasmid YRp12 treated in vitro with ultraviolet- or gamma-radiation, or with restriction endonucleases, has been used to investigate in vivo RAD gene activity in Saccharomyces cerevisiae. Yields of pyrimidine dimers or single and double strand breaks in plasmid DNA were assayed by physical methods. The biological effects of these damages were assayed by transformation of wild-type cells and rad mutants from each of the major groups of radiosensitive mutants. After UV-irradiation plasmid survival depended qualitatively on the same host functions that are needed for cellular survival. After gamma-irradiation no such correspondence was found. Apart from a RAD52-dependent stimulation of transformation efficiency at low doses, other host repair functions had little effect. Stimulation of transformation corresponded with the production of double- but not single-strand breaks in plasmid sequences homologous with the yeast genome and may be linked with a transient increase in mitotic stability. More generally these data also show that transformation events using the LiCl protocol may entail the uptake of a very low number of plasmid molecules per cell over a 10-fold range of DNA concentrations.