The Arabidopsis MIM gene encodes a protein belonging to the SMC family (structure maintenance of chromosomes) which is required for intrachromosomal homologous recombination (ICR). Both ICR and MIM gene expression are enhanced by DNA-damaging treatments, suggesting that MIM is a factor limiting DNA repair by homologous recombination (HR) under genotoxic stress. We tested this hypothesis by measuring the levels of recombination in the mim mutant under genotoxic stress, using methyl methanesulfonate. Although the mutant clearly showed diminished basal and induced levels of ICR, enhancement of ICR by DNA-damaging treatments was similar to that observed in the wild type. This suggests that the MIM gene product is required for DNA repair by HR, but is not critical for HR induction. To determine whether enhanced availability of MIM would increase basal HR levels in Arabidopsis, we examined ICR frequencies in transgenic Arabidopsis strains overexpressing the MIM gene after ectopic insertion of additional MIM copies. Two independent lines showed a twofold increase in ICR frequency relative to the wild type. Thus MIM is required for efficient ICR in plants, and its manipulation can be used to change homologous recombination frequencies. Since MIM is one of the components responsible for chromatin dynamics, our results suggest that the chromatin environment determines the frequency of homologous recombination.

Introduction

DNA double-strand breaks (DSB) can be produced by ionizing radiation or genotoxic chemicals, and may also occur during DNA replication. Unrepaired lesions have severe consequences, such as inhibition of DNA replication, partial or entire loss of chromosomes, and cell death. There are two competing classes of mechanisms by which DSB can be repaired: homologous recombination (HR), and non-homologous end joining (NHEJ) (for reviews see Caroll, 1996; Haber, 1999; Kanaar et al., 1998 ; Osman and Subramani, 1998). HR is the major pathway in prokaryotes and yeast, whereas NHEJ predominates in mammals and plants. Although NHEJ is the major pathway, HR still makes a significant contribution, for example in mammalian cells ( Liang et al., 1998 ). NHEJ in mammalian cells is initiated by the DNA-dependent protein kinase (DNA-PK), which consists of several subunits. The core serine/threonine kinase, encoded by the scid gene ( Blunt et al., 1995 ), is associated with the Ku antigen, a heterodimer of 70 and 80 kDa subunits that bind to broken DNA ends ( Mirmori et al., 1981 ). Ku proteins are present in vertebrates, insects, worms and yeast (for review see Tuteja and Tuteja, 2000); however, Ku-like proteins have not yet been identified in plants. Ku, associated with DSB, recruits DNA-PK. The DNA/Ku complex is activated by autophosphorylation at Ku ( Rathmell and Gilbert, 1998), and the repair can then be initiated by processing of broken ends and their ligation. NHEJ often leads to deletions or insertions of filler DNA fragments at the lesion site, which has also been observed in plants ( Salomon and Puchta, 1998). HR makes use of a DNA template homologous to the damaged molecule to precisely repair the lesion ( Shinohara and Ogawa, 1995). Interest in regulatory aspects of the two pathways and their mechanisms has been enlivened by the importance of homologous recombination in gene-targeting technology. In flowering plants, gene targeting is complicated by the low frequencies of HR relative to NHEJ.

Several approaches have been taken to increase HR. It has been shown, for example, that intrachromosomal homologous recombination (ICR) can be induced by elevated DNA damage ( Lebel et al., 1993 ; Puchta et al., 1995 ), or by the introduction of DSB by endonucleolytic cleavage ( Puchta et al., 1996 ). In Saccharomyces cerevisiae the DUN1 serine/threonine kinase, in cooperation with RAD53 gene product, is required for transcriptional induction of genes involved in damage-induced repair ( Allen et al., 1994 ; Elledge et al., 1993 ; Zhou and Elledge, 1993). In plants the molecular mechanism controlling cellular responses to increased DNA damage is still unknown, although transcriptional induction of recombination-related genes has been observed ( Doutriaux et al., 1998 ; Lebel et al., 1993 ; Mengiste et al., 1999 ; Puchta et al., 1995 ). Thus increased DNA damage initiates signals that promote recombinational repair, but it is likely that both HR and NHEJ are activated.

A further approach to stimulating HR in plants involves transgenic expression of bacterial proteins with well defined functions in the prokaryotic recombination process. RecA or resolvase RuvC introduced into tobacco plants led to an increase in ICR ( Reiss et al., 1996 ; Shalev et al., 1999 ), suggesting that these activities from distant species can co-operate with plant recombination complexes. As the nature of these complexes, and their role in HR, are unclear, it is difficult to define the mechanism by which foreign proteins stimulate HR in plants.

Recently we have identified an Arabidopsis mutant (mim) impaired in intrachromosomal homologous recombination in somatic cells ( Mengiste et al., 1999 ). MIM encodes an SMC-like protein (structure maintenance of chromosomes). This conserved SMC family of chromatin-associated proteins (for reviews see Hirano, 1998; Jessberger et al., 1998 ; Strunnikov, 1998) has a large spectrum of chromosome-related activities such as sister chromatid cohesion ( Guacci et al., 1997 ; Michaelis et al., 1997 ); gene dosage compensation ( Lieb et al., 1998 ); and DNA recombination ( Jessberger et al., 1996 ; Lehmann et al., 1995 ; Mengiste et al., 1999 ). The MIM protein shares the highest homology with S. pombe RAD18 protein. Although the rad18 null mutation is lethal, indicative of additional functions in Schizosaccharomyces pombe ( Fousteri and Lehmann, 2000), the temperature-sensitive rad18 mutation is epistatic to the rhp51 gene involved in homologous recombination ( Lehmann et al., 1995 ). In contrast, inactivation of the Arabidopsis MIM gene results in a phenotype restricted to increased sensitivity to genotoxic treatments. This is accompanied by a significant reduction in the frequency of ICR ( Mengiste et al., 1999 ), which suggests a more specialized function of MIM in securing basal recombination levels. It was also postulated that MIM can be involved in post-replication recombinational repair, as the mim mutant shows slower root growth during germination, and sensitivity to elevated temperature ( Mengiste et al., 1999 ). Since MIM expression was shown to be induced by methyl methanesulfonate (MMS), which also stimulates ICR in plants, we examined the influence of this gene on induction of ICR by MMS. It is most likely that MIM acts at the level of chromatin structure, probably by facilitating access of repair complexes to the damage site. Thus we investigated whether an increase in MIM stimulates ICR. Enhancement of ICR due to MIM overexpression would not only demonstrate that it is possible to stimulate HR using the native plant protein, but would also support the possibility that chromatin structure is a critical factor limiting HR efficiency in plants

Results

Induction of intrachromosomal homologous recombination is MIM independent

MMS highly stimulates ICR in Arabidopsis ( Puchta et al., 1995 ) and also induces MIM gene expression ( Mengiste et al., 1999 ). To determine whether activity of the MIM gene is required for induction of HR, we compared ICR frequencies after MMS induction in wild-type plants and mim mutant. The recombination assay was performed using a transgenic recombination trap of line N1DC1 No. 11 (L11), consisting of direct repeats of overlapping parts of a chimeric β-glucuronidase (GUS) gene ( Swoboda et al., 1994 ). Since HR within repeats of the GUS gene restores both structure and function, the number of blue sectors detected after histochemical staining directly indicates HR frequency. We generated F₃ populations after crossing the mim mutant with L11 and identified the following genotypes: mim/mim, L11/L11 and MIM/MIM, L11/L11 ( Mengiste et al., 1999 ). Using these genotypes we compared ICR frequencies in the absence or presence of MMS at a concentration optimal for ICR induction (40 p.p.m.) but not harmful for development. ICR frequency in the mim/mim L11/L11 background was reduced in both control and MMS-containing media, in comparison to MIM/MIM, L11/L11 ( Table 1). The reduction of HR in the control medium was lower than previously described by Mengiste et al. (1999) , which may be due to different growth conditions (see Experimental procedures). Surprisingly, ICR induction in MIM/MIM L11/L11 and in mim/mim L11/L11 populations was similar (6.8- and 5.36-fold, respectively). Thus the presence of MIM is required for ICR under standard growth conditions and under genotoxic stress, but its absence does not prevent ICR induction.

Table 1. Induction of homologous recombination by MMS in mim and wild-type plants

Genotypes	MMS (p.p.m.)	Number of plants tested	Recombination frequencies ^a	Degree of induction
mim/mim, L11/L11	0	49	0.76
mim/mim, L11/L11	40	41	4.08	5.36
MIM/MIM, L11/L11	0	48	1.47
MIM/MIM, L11/L11	40	41	9.75	6.8

^a Recombination frequencies are mean values of two GUS assays, each carried out on 15–28 seedlings (3 weeks old) of both genotypes. Frequencies were calculated by dividing the total number of recombination events by the number of plants tested.

Generation and characterization of Arabidopsis lines overexpressing MIM

Although MIM function is not a prerequisite for induced recombination following DNA damage, its absence causes hypersensitivity to genotoxic stress and a significant general reduction in HR in the ICR assay ( Mengiste et al., 1999 ). Thus it was interesting to examine whether an increase in MIM expression would result in both higher genotoxic stress resistance and enhanced HR. In the course of mutant complementation with the genomic wild-type gene, several transgenic lines regained resistance to MMS. Northern blots with the 5′ part of MIM cDNA as a probe revealed highly variable MIM mRNA levels among these lines ( Figure 1). Seedlings of three complemented lines (L1, L2 and L7) had MIM transcripts at approximately the wild-type level. However, two further lines (L8 and L9) showed, on average, a sevenfold increase of MIM transcript, reaching a MIM mRNA abundance higher than that in a rapidly dividing suspension culture of Arabidopsis ( Figure 1). In the hybrid L9 × L11, used for determination of ICR frequencies, the level of MIM mRNA is increased. As deduced from Southern blot analysis ( Figure 2), each of these two overexpressing lines has a single T-DNA insert, suggesting that overexpression is due to the position of the transgenic inserts, or that these plants constitutively activate the MIM gene. To distinguish between these possibilities, expression of the Arabidopsis RAD51 homologue, known to be induced by genotoxic stress ( Doutriaux et al., 1998 ), was examined. This transcript was at the wild-type level in all transgenic lines (data not shown), indicating that the transgene insertion sites in line L8 or L9 are responsible for MIM overexpression. L8 and L9 are phenotypically indistinguishable from wild-type plants, and therefore MIM overexpression does not seem to affect plant development.

Details are in the caption following the image — **Figure 1**
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Northern blot analysis of total RNA (15 μg) extracted from seedlings of wild-type *Arabidopsis* Wassilewskija (WS), *MIM* complemented lines (L1, L2, L7, L8 and L9), hybrids (L9 × L11 and WS × L11), and suspension culture cells (SC).

The DNA probe was a 1 kb *Eco*RI/*Xho*I fragment of the 5′ part of the cDNA ( Mengiste *et al*., 1999 ). The blot was rehybridized to probe of the constitutively expressed *RAN* gene ( Haizel *et al*., 1995 ). The radioactive bands were quantified using a Storm860 phosphorimager.

MMS sensitivity

Since mim is hypersensitive to DNA-damaging treatments (MMS, X-rays, UV light; Mengiste et al., 1999 ), we examined whether overexpression of the MIM gene in L8 and L9 results in plants highly resistant to genotoxic stress. F₃ seedlings of L8 and L9 homozygous for the ectopic MIM copy were transferred, together with the wild type and the mutant, to multi-well plates containing germination medium with MMS at increasing concentrations. While 100 p.p.m. MMS was lethal to mim seedlings, the wild-type seedlings tolerated 125 p.p.m. ( Figure 3). However, neither L9 ( Figure 3) nor L8 (data not shown) was more MMS resistant than the wild type.

MIM overexpression and intrachromosomal homologous recombination

To examine levels of ICR in the L8 and L9 backgrounds, we constructed F₁ hybrids containing both transgenic loci, which thus overexpressed the MIM gene and contained the N1DC1 No. 11 GUS recombination substrate (L11). The hybrid seedlings L9 × L11 or L8 × L11, and control WS × L11 seedlings (F₁ hybrid between wild-type Wassilewskija and L11), were harvested and stained histochemically for GUS activity. The mean ICR frequency in L8 × L11 and L9 × L11 hybrids exceeded by 1.8-fold and twofold, respectively, that of the control ( Table 2). The absolute ICR frequencies in MIM/MIM, L11/L11 ( Table 1) and in WS × L11 ( Table 2) cannot be compared, and this is due to different experimental conditions.

Table 2. Effect of MIM overexpression on ICR

Hybrids	Number of plants tested	Number of recombination events	Recombination frequencies ^a	Degree of induction
WS × L11 (F₁)	31	448	14.45
L8 × L11(F₁)	32	836	26.13	1.80
L9 × L11 (F₁)	32	953	29.78	2.06

^a Recombination frequencies are means of three values for 10–11 seedlings (5 weeks old) chosen according to developmental stage. Frequencies were calculated by dividing the total number of recombination events by the number of plants tested.

ICR induction due to MIM overexpression also became obvious in comparisons of the distribution of recombination events ( Figure 4). In the control, 50% of seedlings exhibited a maximum of 10 recombination events per plant, whereas 90% of L9 × L11 seedlings showed more than 10 events per plant; more than half of the latter had frequencies above 20 events per plant. Therefore MIM is a factor limiting homologous recombination between GUS repeats integrated in Arabidopsis, and overexpression of the MIM gene significantly increases ICR in somatic cells.

Discussion

The discovery of factors influencing levels of HR in higher plants, and studies of their functions, will increase our understanding of the process and the possibilities for its control. Here we describe the first plant element (MIM) required for achieving optimal recombination levels in Arabidopsis somatic cells under normal growth conditions and following genotoxic stress. The MIM gene was isolated in a search for mutations affecting sensitivity to genotoxic stress. The mim mutant plants have an elevated sensitivity to irradiation with UV-C and X-rays, and to the DNA-damaging agent MMS ( Mengiste et al., 1999 ).

MIM has high similarity to members of the SMC-ATPase family, required for dynamic changes in chromatin architecture ( Jessberger et al., 1998 ). Thus involvement of the MIM gene product in DNA repair is probably not direct, but is rather through modification of chromatin in the vicinity of a damaged site which facilitates access of repair complexes. Interestingly, complete depletion of MIM is not only permissive, but mim mutant plants also have an unaltered morphology under standard growth conditions ( Mengiste et al., 1999 ). They show significant reduction in ICR in somatic tissues, but are still able to carry out the process. This suggests that MIM can be replaced by another unknown protein(s), or that its activity facilitates HR repair but is not totally essential. It is apparent, however, that the mim mutant cannot cope with increased DNA damage as successfully as the wild type, as reflected by its hypersensitivity to genotoxic treatments ( Mengiste et al., 1999 ). Challenging plants with low doses of MMS which are harmless for growth of both the mim mutant and the wild type, but stimulate HR by increasing demand for DSB repair, resulted in increases in ICR frequencies in both wild-type and mim plants. Thus the signalling leading to ICR induction appears to be unaffected in the mutant. However, the absolute frequencies of intrachromosomal recombination in mim plants were lower than in the wild type under both conditions. In S. cerevisiae, several rad mutations also reduce spontaneous, as well as γ-ray-induced, mitotic recombination measured as interchromosomal recombination ( Saeki et al., 1980 ). The genes concerned (such as RAD55 and RAD57) belong to the RAD52 epistasis group which is implicated in the recombinational repair of DSB. Taking into consideration the clearly different lethal levels of genotoxic stress between the mutant and the wild type, it could be envisaged that MIM function is essential for plant survival above a particular damage level. It is, however, conceivable that the degree of DNA damage under standard growth conditions or under low genotoxic stress is similar for the wild type and mim. In this case, the most plausible explanation for successful repair in the mutant which results in unaltered growth characteristics is increased utilization of another repair pathway (e.g. NHEJ) to compensate for the lack of the MIM function required for ICR. NHEJ could not be measured in our experimental setup, but the patterns of T-DNA integration in mim and wild-type plants are comparable, with one or more T-DNA insertions in individual transformants (data not shown). Thus MIM does not appear to interfere with NHEJ. This suggests that MIM expression can influence the choice of DSB repair between HR and other alternatives.

Consequently, overexpression of the MIM gene should favour ICR. We identified two independent Arabidopsis lines (L9 and L8) which had a significant elevation of MIM transcripts. Each contained a single T-DNA insert carrying the MIM gene. The transgenes span the genomic region of MIM, including 2 kb of the promoter region and 0.5 kb of the 3′ portion beyond the coding sequence. The up-regulation of this transcript is probably due to the particular chromosomal positions of the transgene, as other complemented lines (such as L1) show a very low level of transcript. However, MIM overexpression did not lead to an increase in MMS resistance. Lines with either high or low MIM expression reverted to the wild-type tolerance to MMS. Thus for the wild-type resistance to MMS genotoxic stress, the abundance of the MIM transcript is not a decisive factor. It is likely that other components contribute equally to genotoxic stress tolerance by Arabidopsis.

The ICR frequencies in F₁ hybrids of L9 × L11 and L8 × L11 were approximately twofold higher than in the wild-type control cross. Importantly, a similar level of HR induction was observed in these two independent lines, with different integration sites for the T-DNA carrying the MIM locus. Furthermore, the increased HR frequencies were accompanied by a change in the distribution of recombination events within the plant population. Overexpression of MIM led to a higher proportion of plants with an elevated number of recombination events. The increase in HR in lines overexpressing MIM is in agreement with the hypothesis that MIM supports a preference for HR over NHEJ in DSB repair. Competitive binding to DSB, by either the Rad52 protein or Ku, was proposed to direct DSB repair towards HR or NHEJ, respectively ( Van Dyck et al., 1999 ). Although Ku and Rad52 homologous proteins have not yet been identified in plants, it can be envisaged, in the chromatin context, that the competition between the two classes of repair mechanisms is influenced by selective DNA accessibility through modification of chromatin structure. MIM, an SMC-like protein, may be involved in modification of chromatin structure, allowing entry of HR but not other repair complexes.

Our results represent a first step towards regulation of HR in plants using endogenous factors. The further characterization of plant DNA repair components will substantially contribute towards better control over homologous recombination in this group of organisms.

Experimental procedures

Arabidopsis growth conditions

Seeds of Wassilewskija ecotype were plated on germination medium and kept for 2 days at 4°C. The plates were then transferred to a growth chamber with 16 h light, 25 μE m⁻² s⁻¹ (Osram Natura) at 23°C, 8 h dark at 16°C.

MMS sensitivity tests

MMS sensitivity tests on 4-week-old seedlings of wild-type, mim and overexpressing lines (L9 and L8), all in Wassilewskija background, were performed at 0–200 p.p.m. as described previously ( Masson et al., 1997 ).

Intrachromosomal recombination assay (ICR)

Five-week-old seedlings of L9 × L11, L8 × L11 and WS × L11 hybrids were subjected to GUS histochemical staining using X-glu, as described previously ( Jefferson et al., 1987 ). For induction of ICR by MMS, 2-week-old seedlings of mim/mim, L11/L11 and MIM/MIM, L11/L11 lines were transferred to liquid germination medium with MMS at 40 p.p.m. or without MMS, and incubated for 1 week before GUS histochemical staining.

Northern and Southern blot analysis

DNA was extracted from 3-week-old seedlings as described by Dellaporta et al. (1983) ; digested with restriction endonucleases as indicated, and separated electrophoretically. For Southern blotting, DNA fragments were transferred for 1 h to nylon membranes (Hybond N, Amersham Pharmacia Biotech, Amersham, Buckinghamshire, UK) using the POSIBLOT 30–30 Blotter (Stratagene, La Jolla, CA, USA) according to the manufacturer's instructions. After transfer, the membranes were washed briefly with transfer buffer and then crosslinked (1200 kj cm⁻²) using a Stratalinker apparatus (Stratagene, La Jolla, CA, USA).

RNA was extracted from 3-week-old seedlings using TRIZOL reagent (Gibco BRL, Grand Island, NY, USA) according to the supplier's instructions. RNA was transferred to nylon membranes using standard protocols ( Sambrook et al., 1989 ). Hybridization of Southern and Northern blots was performed as described ( Church and Gilbert, 1984).

Acknowledgements

We would like to thank Karin Afsar for technical assistance, and Ortrun Mittelsten Scheid, Patrick King and Barbara Hohn for critically reading the manuscript. We are also grateful to Novartis Research Foundation. This work was supported by the Swiss Federal Office for Education and Science (BBW Grant No. 96 0402-1) and the European Union (EU Grant No. B104-C797-2028).

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Citing Literature

Volume24, Issue2

October 2000

Pages 183-189

Elevated levels of intrachromosomal homologous recombination in Arabidopsis overexpressing the MIM gene

Summary

Introduction

Results

Induction of intrachromosomal homologous recombination is MIM independent