SummaryThe Fanconi anemia (FA) core complex promotes the tolerance/repair of DNA damage at stalled replication forks by catalyzing the monoubiquitination of FANCD2 and FANCI. Intriguingly, the core complex component FANCM also catalyzes branch migration of model Holliday junctions and replication forks in vitro. Here we have characterized the ortholog of FANCM in fission yeast Fml1 in order to understand the physiological significance of this activity. We show that Fml1 has at least two roles in homologous recombination—it promotes Rad51-dependent gene conversion at stalled/blocked replication forks and limits crossing over during mitotic double-strand break repair. In vitro Fml1 catalyzes both replication fork reversal and D loop disruption, indicating possible mechanisms by which it can fulfill its pro- and antirecombinogenic roles.

IntroductionCells of sexually reproducing eukaryotes normally contain two equal (homologous) sets of chromosomes, one contributed by the father and the other by the mother during the fusion of gametes and the formation of a zygote. Meiosis is the cell division that reduces the number of chromosomes by half. It produces gametes or their precursor cells, each of which contains a haploid set consisting of randomly assorted parental chromosomes. These chromosomes are mosaics, because the original parental homologues have exchanged corresponding pieces by crossing-over. Thus, the function of meiosis is twofold -it compensates for the doubling of the chromosome number at fertilization and it provides the progeny with newly assorted sets of alleles, which is the basis of their genetic diversity.Crossing-over is initiated at multiple sites in recombining chromosomes by the enzymatic induction of double-strand breaks (DSBs). The resection of single strands at DSBs leads to the formation of gaps in the DNA. The missing bases are replenished by using the complementary sequence from the homologous chromosome as the template. This process is recombinogenic; that is, it can lead to the reciprocal exchange of DNA between the chromosomes involved (for review, see Keeney, 2001).In order to allow crossing-over, homologous parental chromosomes must pair during meiotic prophase via a ladderlike proteinaceous structure, the synaptonemal complex (SC) (for reviews, see Loidl, 1990;Zickler and Kleckner, 1999). The SC consists of two parallel axes (the lateral elements), to each of which the two chromatin threads of a single replicated chromosome are attached. The lateral elements are connected and kept at a distance of ~100 nm by the so-called transversal filaments. The ultrastructure of the SC is evolutionarily well conserved from protists to humans, although its molecular composition is far more heterogeneous.The fission yeast Schizosaccharomyces pombe features a meiosis that is unique in several respects. Most remarkably, it lacks an SC. Instead, so-called linear elements (LEs) appear during meiotic prophase (Olson et al., 1978;Bähler et al., 1993). LEs appear in the electron microscope (EM) as single lines of variable length, networks of interconnected lines or bundles of lines. These different morphological classes were found to prevail at different stages of meiotic prophase (Bähler et al., 1993), which suggests that their change in appearance is functionally related to chromosome pairing and/or recombination.Because a rec10 mutant lacks LEs, a structural or regulatory role of the Rec10 protein in LE formation has been proposed (Molnar et al., 2003). Apart from this indirect evidence, information on the molecular composition of LEs is scarce. Neither topoisomerase II nor Rec8 [which, in other organisms, constitute the cores along which lateral elements form (Klein et al., 1992;Klein et al., 1999)] delineate entire LEs (Hartsuiker et al., 1998;Parisi et al., 1999;Watanabe and Nurse, 1999), and S. pombe homologues of proteins prese...

The formation of healthy gametes depends on programmed DNA double strand breaks (DSBs), which are each repaired as a crossover (CO) or non-crossover (NCO) from a homologous template. Although most of these DSBs are repaired without giving COs, little is known about the genetic requirements of NCO-specific recombination. We show that Fml1, the Fanconi anemia complementation group M (FANCM)-ortholog of Schizosaccharomyces pombe, directs the formation of NCOs during meiosis in competition with the Mus81-dependent pro-CO pathway. We also define the Rad51/Dmc1-mediator Swi5-Sfr1 as a major determinant in biasing the recombination process in favour of Mus81, to ensure the appropriate amount of COs to guide meiotic chromosome segregation. The conservation of these proteins from yeast to Humans suggests that this interplay may be a general feature of meiotic recombination. KeywordsHomologous recombination; Meiosis; Fml1; Mus81; Schizosaccharomyces pombe Faithful chromosome segregation during meiosis depends on the establishment of chiasmata through recombinational repair of programmed DNA double-strand breaks (DSBs) to produce crossovers (COs) between homologous chromosomes (homologs). However, in most cases only a minority of the DSBs are earmarked to form COs, and therefore the majority have to be repaired by using either the homolog without CO formation or the sister chromatid (1).In order to identify helicase activities involved in non-crossover (NCO)-recombination during meiosis in the fission yeast Schizosaccharomyces pombe, we screened for helicases potentially capable of D loop unwinding during synthesis-dependent strand annealing (SDSA), which is thought to be a major pathway of NCO recombination (1). To this end, we used a genetic recombination assay consisting of a meiotic recombination hotspot at the ade6 gene and two flanking scorable markers (Fig. 1A). We hypothesized that at least one of the helicases promoting NCO recombination pathways in mitotic cells would also have a role during meiosis. From our candidate list -fbh1, srs2, rqh1, fml1 and fml2 -only the deletion of fml1 gave the expected increase in CO formation associated with a meiotic gene Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts conversion (GC) event at two different hotspot alleles, ade6-M26 and ade6-3083, and at a non-hotspot allele ade6-M375 (Fig. 1, B and C, and tables S1 to S3) (2-5). Increases in COs were also observed on a different chromosome (Fig. 1D and table S4) and by a physical assay at the mbs1 locus ( fig. S1), indicating that Fml1's role in suppressing CO formation is not restricted to a single locus.In vitro purified Fml1, like its budding yeast ortholog Mph1, unwinds D loops and is therefore suited to promoting SDSA ( Fig. 1E) (6, 7). The fml1-K99R mutant, which encodes protein that retains full DNA binding activity but is unable to unwind D loops ( Fig. 1E and fig. S2), exhibits the same hyper-CO phenotype as the null mutant indicating that Fml1's helicase function is required for NCO...

Little is known about what dictates the round shape of the yeast Saccharomyces cerevisiae nucleus. In spo7⌬ mutants, the nucleus is misshapen, exhibiting a single protrusion. The Spo7 protein is part of a phosphatase complex that represses phospholipid biosynthesis. Here, we report that the nuclear protrusion of spo7⌬ mutants colocalizes with the nucleolus, whereas the nuclear compartment containing the bulk of the DNA is unaffected. Using strains in which the nucleolus is not intimately associated with the nuclear envelope, we show that the single nuclear protrusion of spo7⌬ mutants is not a result of nucleolar expansion, but rather a property of the nuclear membrane. We found that in spo7⌬ mutants the peripheral endoplasmic reticulum (ER) membrane was also expanded. Because the nuclear membrane and the ER are contiguous, this finding indicates that in spo7⌬ mutants all ER membranes, with the exception of the membrane surrounding the bulk of the DNA, undergo expansion. Our results suggest that the nuclear envelope has distinct domains that differ in their ability to resist membrane expansion in response to increased phospholipid biosynthesis. We further propose that in budding yeast there is a mechanism, or structure, that restricts nuclear membrane expansion around the bulk of the DNA. INTRODUCTIONThe nucleus has a distinct organization, characterized by the presence of internal subcompartments. Moreover, in most, but not all, cell types the nucleus adopts a round shape. Understanding how this organization and shape are achieved is of major biological and medical interest. Certain cell types, such as those found in blood lineages (e.g., neutrophils, monocytes, and eosinophils), undergo dramatic nuclear shape changes as they differentiate (Gartner et al., 1990). Cancerous states are often associated with changes in nuclear morphology, most frequently nucleolar enlargement, changes in nuclear shape, or a combination of the two (Zink et al., 2004). Although these changes provide useful diagnostic markers of cancer progression, their mechanistic basis is still poorly understood. Other diseases are also associated with changes in nuclear shape and organization. For example, Hutchinson-Gilford progeria syndrome, which leads to premature aging, is caused by a specific mutation in the gene encoding A-type lamins and is associated with nuclear shape changes . Interestingly, progeria-like changes in nuclear shape are part of the normal aging process of nonneuronal cells in Caenorhabditis elegans (Haithcock et al., 2005). Genetic defects in the nuclear lamina are also associated with several types of muscular dystrophy . Nuclear lamins and their associated proteins provide both a rigid structure that helps shape the nuclear membrane and a platform onto which protein complexes and chromatin can bind (Holaska et al., 2002). Although much has been learned in recent years about the function of nuclear lamins, many significant questions remain. For example, it is not known how diseaseassociated changes in nuclear shape affect...

In fission yeast, meiotic prophase nuclei develop structures known as linear elements (LinEs), instead of a canonical synaptonemal complex. LinEs contain Rec10 protein. While Rec10 is essential for meiotic recombination, the precise role of LinEs in this process is unknown. Using in situ immunostaining, we show that Rec7 (which is required for meiosis-specific DNA double-strand break (DSB) formation) aggregates in foci on LinEs. The strand exchange protein Rad51, which is known to mark the sites of DSBs, also localizes to LinEs, although to a lesser degree. The number of Rec7 foci corresponds well with the average number of genetic recombination events per meiosis suggesting that Rec7 marks the sites of recombination. Rec7 and Rad51 foci do not co-localize, presumably because they act sequentially on recombination sites. The localization of Rec7 is dependent on Rec10 but independent of the DSB-inducing protein Rec12/Spo11. Neither Rec7 nor Rad51 localization depends on the LinE-associated proteins Hop1 and Mek1, but the formation of Rad51 foci depends on Rec10, Rec7, and, as expected, Rec12/Spo11. We propose that LinEs form around designated recombination sites before the induction of DSBs and that most, if not all, meiotic recombination initiates within the setting provided by LinEs.