Control of meiotic recombination in Arabidopsis ... - Semantic Scholar

3 downloads 0 Views 197KB Size Report
MutS homologues MSH4 and MSH5 together with the MutL homologues MLH3 and MLH1 are crucial for the formation of meiotic crossovers, but details remain ...
542

Biochemical Society Transactions (2006) Volume 34, part 4

Control of meiotic recombination in Arabidopsis: role of the MutL and MutS homologues F.C.H. Franklin1 , J.D. Higgins, E. Sanchez-Moran, S.J. Armstrong, K.E. Osman, N. Jackson2 and G.H. Jones The School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K.

Abstract Immunocytochemistry reveals that the Arabidopsis mismatch repair proteins AtMSH4, AtMLH3 and AtMLH1 are expressed during prophase I of meiosis. Expression of AtMSH4 precedes AtMLH3 and AtMLH1 which colocalize as foci during pachytene. Co-localization between AtMSH4 and AtMLH3 occurs, but appears transient. AtMLH3 foci are not detected in an Atmsh4 mutant. However, localization of AtMSH4 is unaffected in Atmlh3, suggesting that recombination may proceed to dHj (double Holliday junction) formation. Mean chiasma frequency in Atmsh4 is reduced to 1.55 compared with 9.86 in wild-type. In contrast with wild-type, the distribution of residual crossovers in Atmsh4 closely fits a Poisson distribution. This is consistent with a twopathway model for meiotic crossing-over whereby most crossovers occur via an AtMSH4-dependent pathway that is subject to interference, with the remaining crossovers arising via an interference-independent pathway. Loss of AtMLH3 results in an approx. 60% reduction in crossovers. Results suggest that dHj resolution can occur, but in contrast with wild-type where most or all dHjs are directed to form crossovers, the outcome is biased in favour of a non-crossover outcome. The results are compatible with a model whereby the MutL complex maintains or imposes a dHj conformation that ensures crossover formation.

Introduction Meiosis is a specialized form of cell division during which a single round of DNA replication is followed by two cell divisions thereby reducing the chromosome content from diploid to haploid. Accurate segregation of homologous chromosomes at the first meiotic division is dependent on the formation of physical connections, known as chiasmata, between homologous chromosome pairs (homologues). Chiasmata arise from HR (homologous recombination) during prophase I of meiosis and are the physical manifestation of genetic crossovers. In their absence, the homologues segregate at random, leading to the formation of aneuploid gametes after the separation of the sister chromatids at the second meiotic division. Eukaryotic organisms possess multiple homologues of the bacterial MMR (mismatch repair) proteins MutS and MutL [1,2]. The eukaryotic MMR proteins are essential for maintaining genome integrity during mitosis and meiosis. However, several MMR proteins are also required for HR during prophase I of meiosis. Previous studies have revealed that the MutS homologues MSH4 and MSH5 together with the MutL homologues MLH3 and MLH1 are crucial for the formation of meiotic crossovers, but details remain unclear [1–4]. To provide further insight into the meiotic function of the MMR proteins, we have investigated the role of AtMSH4, AtMLH3

Key words: Arabidopsis, crossover interference, double Holliday junction, homologous recombination, meiosis, mismatch repair. Abbreviations used: dHj, double Holliday junction; HR, homologous recombination; MMR, mismatch repair; RI, recombination intermediate. 1 To whom correspondence should be addressed (email [email protected]). 2

Present address: Centre for Genetics and Development, Section of Microbiology and Section of Molecular and Cellular Biology, University of California, Davis, CA 95616, U.S.A.

 C 2006

Biochemical Society

and AtMLH1 during meiosis in the plant Arabidopsis thaliana [5,6].

Distribution of AtMSH4, AtMLH3 and AtMLH1 proteins during prophase I The distribution and chronology of the MMR proteins in chromosome spread preparations of meiocytes at different stages of prophase I were investigated using fluorescence immunolocalization (Figures 1A–1F) [5,6]. This revealed that foci corresponding to AtMSH4 first appear in association with the chromosome axes at leptotene. Initially, the foci were numerous with approx. 100 per nucleus. As prophase I progressed through zygotene into pachytene, the number of foci continuously decreased such that by late pachytene the AtMSH4 signal was absent (Figures 1A–1C). AtMLH3 foci were first detectable during zygotene, gradually increasing to a mean number of 9.4 per nucleus, which closely corresponds to the number of chiasmata detected at metaphase I (Figures 1D and 1E) [6]. Normal localization of AtMLH3 was dependent on AtMSH4, but colocalization of the proteins appeared to be transient since we consistently observed only 1–2 dual foci per nucleus. During pachytene, AtMLH3 was found to co-localize with AtMLH1 (Figure 1F). In an Atmlh3 mutant, AtMLH1 pachytene foci were absent and the protein was found to accumulate in the nucleolus (Figure 1G), whereas the distribution and turnover of AtMSH4 were indistinguishable from wild-type.

Crossover distribution in an Atmsh4 mutant indicates two meiotic recombination pathways Analysis of T-DNA (transfer DNA) insertion lines Atmsh4 and Atmlh3 revealed that loss of either protein had no

Meiosis and the Causes and Consequences of Recombination

Figure 1 Localization of MMR proteins during prophase I and cytological analysis of chiasma frequency in MMR mutants (A–C) Immunolocalization of AtMSH4 (red) in spread preparations of wild-type nuclei during prophase I. (A) leptotene; (B) zygotene; (C) early pachytene. (D, E) Immunolocalization of AtMLH3 (red) in wild-type nuclei during prophase I (images collected as Z-stacks). (D) Early zygotene; (E) late zygotene. (F) Co-localization of AtMLH3 and AtMLH1 (yellow) at late pachytene in wild-type nuclei. (G) AtMLH1 (red) accumulates in the nucleolar region in an Atmlh3 mutant. Chromosome axes are labelled with anti-ASY1 (meiotic asynaptic mutant 1) antibody (green) throughout. Scale bar, 10 µm. (H–J) Examples of metaphase I nuclei revealing reduced chiasma frequency in Atmsh4 and Atmlh3 mutants. Fluorescence in situ hybridization probes recognizing 5 S rDNA (recombinant DNA) (red) and 45 S rDNA (green) allow the identification of each bivalent chromosome. (H) Typical wild-type nucleus; (I) Atmlh3 nucleus with four chiasmata (chromosomes 1, 3, 4 and 5); (J) Atmsh4 nucleus with two chiasmata (chromosomes 4 and 5). Scale bar, 10 µm.

apparent effect on early recombination events in early prophase I. However, as the chromosomes desynapsed towards the end of prophase I, it became clear that a proportion of the homologues lacked chiasmata. Examination of chiasmata at metaphase I revealed a mean chiasma frequency of 1.55 in Atmsh4 compared with 9.86 in wild-type (Figures 1H and 1J). Despite this low mean, the chiasma numbers per cell were distributed over a wide range of 0–7, although this was markedly skewed with the mode falling in the one-chiasma class. Statistical analysis revealed that the numerical distribution of residual chiasmata per cell and per chromosome did not differ significantly from a Poisson distribution [5]. This random distribution contrasts with that of wild-type which is nonrandom. Studies in budding yeast have led to the proposal that meiotic crossovers occur via two biochemically distinct pathways. Class I crossovers require MSH4/MSH5 and

are subject to crossover interference, the phenomenon that ensures that two crossovers do not occur in adjacent regions along a chromosome. Class II crossovers are proposed to be dependent on MUS81, are randomly distributed and thus do not exhibit interference [7]. In yeast, it is estimated that approx. 15% of crossovers arise independently of the MSH4/MSH5 pathway. This is in close agreement with the residual crossovers observed in the Atmsh4 mutant. Moreover, mathematical modelling of genetically determined crossover distributions in Arabidopsis is compatible with the existence of two crossover pathways [8]. Thus, on the basis of current evidence, it appears that crossovers in Arabidopsis arise via an interference-dependent pathway that accounts for approx. 85% of crossovers and requires AtMSH4 and a second interference-independent pathway that accounts for the remaining crossovers.  C 2006

Biochemical Society

543

544

Biochemical Society Transactions (2006) Volume 34, part 4

Different fates for RIs (recombination intermediates) in AtMSH4- and AtMLH3-deficient backgrounds There is compelling evidence from budding yeast that the MutS and MutL homologues function in the same recombination pathway [3]. Analysis of two independent Atmlh3 mutants revealed a mean chiasma frequency of 3.9 (Figures 1H and 1I) [4,6]. While this represents a substantial reduction compared with wild-type (9.86), it is not as severe as that in Atmsh4. This suggests that the fate of crossoverdesignated RIs is different in the two mutant backgrounds. Further evidence in support of this possibility comes from the use of bromodeoxyuridine pulse-labelling of meiocytes to investigate prophase I progression in the MMR mutants. In wild-type, meiosis is completed in 33 h, whereas an 8 h delay in the first meiotic division occurs in Atmsh4. However, a much greater delay of 25 h is apparent in Atmlh3. One possible explanation arises from biochemical studies using purified human hMSH4/hMSH5 which has led to the proposal that the MutS proteins bind and stabilize progenitor dHjs (double Holliday junctions) [9]. If so, in the absence of AtMSH4, stable dHjs may not be formed, in which case, the Class I RIs would be repaired prior to dHj formation and without the formation of crossovers. In Atmlh3 mutants, dHjs could arise, but their resolution to form crossovers would be compromised. Analysis of the chiasma frequency data from Atmlh3 revealed that they can be fitted to a binomial distribution [6]. This is consistent with a model whereby the crossoverdesignated RIs could resolve in one of two ways, as crossovers or non-crossovers. The best fit value of P (0.35) suggests that approximately one-third of the dHjs may resolve as crossovers in the absence of AtMLH3. No doubt this is an

 C 2006

Biochemical Society

oversimplification because it does not take into account the crossovers that arise from the Class II pathway, but it does indicate that, in the absence of AtMLH3, the default resolution of dHjs to crossovers is too low to ensure a crossover between each homologue pair, which is the minimum requirement to ensure accurate chromosome segregation at the first meiotic division. In summary, our results are compatible with a model whereby AtMSH4 establishes stable dHjs. Subsequently, AtMLH1/AtMLH3 impose or maintain the dHjs in a configuration that ensures their resolution as crossovers by an as yet unidentified resolvase.

Work in the F.C.H.F./G.H.J. laboratory is funded by the Biotechnology and Biological Sciences Research Council, U.K.

References 1 Hoffmann, E.R. and Borts, R.H. (2004) Cytogenet. Genome Res. 107, 232–248 2 Svetlanov, A. and Cohen, P.E. (2004) Exp. Cell Res. 296, 71–79 3 Hunter, N. and Borts, R.H. (1997) Genes Dev. 11, 1573–1582 4 Wang, T.F., Kleckner, N. and Hunter, N. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 13914–13919 5 Higgins, J.D., Armstrong, S.J., Franklin, F.C.H. and Jones, G.H. (2004) Genes Dev. 18, 2557–2570 6 Jackson, N., Sanchez-Moran, E., Buckling, E., Armstrong, S.J., Jones, G.H. and Franklin, F.C.H. (2006) EMBO J. 25, 1315–1323 7 de los Santos, T., Hunter, N., Lee, C., Larkin, B., Loidl, J. and Hollingsworth, N.M. (2003) Genetics 164, 81–94 8 Copenhaver, G.P., Housworth, E.A. and Stahl, F.W. (2002) Genetics 160, 1631–1639 9 Snowden, T., Acharya, S., Butz, C., Berardini, M. and Fishel, R. (2004) Mol. Cell 15, 437–451

Received 27 March 2006