Alexander Lorenz

The FANCM Ortholog Fml1 Promotes Recombination at Stalled Replication Forks and Limits Crossing Over during DNA Double-Strand Break Repair

Summary The 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.

S. pombe meiotic linear elements contain proteins related to synaptonemal complex components

The fission yeast Schizosaccharomyces pombe does not form synaptonemal complexes (SCs) in meiotic prophase nuclei. Instead, thin threads, the so-called linear elements (LEs), are observed at the corresponding stages by electron microscopy. Here, we demonstrate that S. pombe Rec10 is a protein related to the Saccharomyces cerevisiae SC protein Red1 and that it localizes to LEs. Moreover, a homologue to S. cerevisiae Hop1 does exist in S. pombe and we show by in situ immunostaining that it, and the kinase Mek1 (a homologue of which is also known to be associated with SCs), localizes to LEs. These observations indicate the evolutionary relationship of LEs with the lateral elements of SCs and suggest that these structures might exert similar functions in S. cerevisiae and S. pombe.

The Fission Yeast FANCM Ortholog Directs Non-Crossover Recombination During Meiosis

No Crossing Over To ensure the correct division of chromosome during the reduction division of meiosis, homologous chromosomes undergo double-strand breaks that—through crossing over and recombination—link the homologs together (and importantly introduce diversity into the genomes of gametes). But only a minority of these crossovers results in recombination—most are directed into non-crossover pathways. Lorenz et al. (p. 1585), working in the yeast Schizosaccharomyces pombe, and Crismani et al. (p. 1588), working in the higher plant Arabidopsis thaliana, looked for the factors that limit crossovers and promote non-crossover pathways. The homolog of the human Fanconi anemia complementation group M (FANCM) helicase protein was found to be a major meiotic anti-recombinase, which could drive meiotic recombination intermediates into the non-crossover pathway. A homolog of a human Fanconi anemia complementation group protein is involved in controlling crossing over during meiosis. 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 favor 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.

Meiotic recombination proteins localize to linear elements in Schizosaccharomyces pombe

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.

Fbh1 Limits Rad51-Dependent Recombination at Blocked Replication Forks

ABSTRACT Controlling the loading of Rad51 onto DNA is important for governing when and how homologous recombination is used. Here we use a combination of genetic assays and indirect immunofluorescence to show that the F-box DNA helicase (Fbh1) functions in direct opposition to the Rad52 orthologue Rad22 to curb Rad51 loading onto DNA in fission yeast. Surprisingly, this activity is unnecessary for limiting spontaneous direct-repeat recombination. Instead it appears to play an important role in preventing recombination when replication forks are blocked and/or broken. When overexpressed, Fbh1 specifically reduces replication fork block-induced recombination, as well as the number of Rad51 nuclear foci that are induced by replicative stress. These abilities are dependent on its DNA helicase/translocase activity, suggesting that Fbh1 exerts its control on recombination by acting as a Rad51 disruptase. In accord with this, overexpression of Fbh1 also suppresses the high levels of recombinant formation and Rad51 accumulation at a site-specific replication fork barrier in a strain lacking the Rad51 disruptase Srs2. Similarly overexpression of Srs2 suppresses replication fork block-induced gene conversion events in an fbh1Δ mutant, although an inability to suppress deletion events suggests that Fbh1 has a distinct functionality, which is not readily substituted by Srs2.

Ultrafine anaphase bridges, broken DNA and illegitimate recombination induced by a replication fork barrier

Most DNA double-strand breaks (DSBs) in S- and G2-phase cells are repaired accurately by Rad51-dependent sister chromatid recombination. However, a minority give rise to gross chromosome rearrangements (GCRs), which can result in disease/death. What determines whether a DSB is repaired accurately or inaccurately is currently unclear. We provide evidence that suggests that perturbing replication by a non-programmed protein–DNA replication fork barrier results in the persistence of replication intermediates (most likely regions of unreplicated DNA) into mitosis, which results in anaphase bridge formation and ultimately to DNA breakage. However, unlike previously characterised replication-associated DSBs, these breaks are repaired mainly by Rad51-independent processes such as single-strand annealing, and are therefore prone to generate GCRs. These data highlight how a replication-associated DSB can be predisposed to give rise to genome rearrangements in eukaryotes.

The human Holliday junction resolvase GEN1 rescues the meiotic phenotype of a Schizosaccharomyces pombe mus81 mutant

A key step in meiotic recombination involves the nucleolytic resolution of Holliday junctions to generate crossovers. Although the enzyme that performs this function in human cells is presently unknown, recent studies led to the identification of the XPG-family endonuclease GEN1 that promotes Holliday junction resolution in vitro, suggesting that it may perform a related function in vivo. Here, we show that ectopic expression of GEN1 in fission yeast mus81Δ strains results in Holliday junction resolution and crossover formation during meiosis.

The DNA helicase Pfh1 promotes fork merging at replication termination sites to ensure genome stability

Bidirectionally moving DNA replication forks merge at termination sites composed of accidental or programmed DNA-protein barriers. If merging fails, then regions of unreplicated DNA can result in the breakage of DNA during mitosis, which in turn can give rise to genome instability. Despite its importance, little is known about the mechanisms that promote the final stages of fork merging in eukaryotes. Here we show that the Pif1 family DNA helicase Pfh1 plays a dual role in promoting replication fork termination. First, it facilitates replication past DNA-protein barriers, and second, it promotes the merging of replication forks. A failure of these processes in Pfh1-deficient cells results in aberrant chromosome segregation and heightened genome instability.

Roles of Hop1 and Mek1 in Meiotic Chromosome Pairing and Recombination Partner Choice in Schizosaccharomyces pombe

ABSTRACT Synaptonemal complex (SC) proteins Hop1 and Mek1 have been proposed to promote homologous recombination in meiosis of Saccharomyces cerevisiae by establishment of a barrier against sister chromatid recombination. Therefore, it is interesting to know whether the homologous proteins play a similar role in Schizosaccharomyces pombe. Unequal sister chromatid recombination (USCR) was found to be increased in hop1 and mek1 single and double deletion mutants in assays for intrachromosomal recombination (ICR). Meiotic intergenic (crossover) and intragenic (conversion) recombination between homologous chromosomes was reduced. Double-strand break (DSB) levels were also lowered. Notably, deletion of hop1 restored DSB repair in rad50S meiosis. This may indicate altered DSB repair kinetics in hop1 and mek1 deletion strains. A hypothesis is advanced proposing transient inhibition of DSB processing by Hop1 and Mek1 and thus providing more time for repair by interaction with the homologous chromosome. Loss of Hop1 and Mek1 would then result in faster repair and more interaction with the sister chromatid. Thus, in S. pombe meiosis, where an excess of sister Holliday junction over homologous Holliday junction formation has been demonstrated, Hop1 and Mek1 possibly enhance homolog interactions to ensure wild-type level of crossover formation rather than inhibiting sister chromatid interactions.

Chromosome Pairing Does Not Contribute to Nuclear Architecture in Vegetative Yeast Cells

ABSTRACT There are several reports of a closer-than-random colocalization of homologous chromosomes in the vegetative nuclei of diploid budding yeast. Here, we studied by fluorescence in situ hybridization (FISH) the nuclear distribution of chromosomes and found a slight tendency toward closer proximity between homologous (allelic) loci than between any nonhomologous chromosomal regions. We show that most of this preferential association is not due to vegetative (also known as somatic) pairing but is caused by the polar orientation of interphase chromosomes (Rabl orientation). We quantified the occasional loss of detectable fluorescence signals that is inherent to the FISH method. Signal loss leads to the occurrence of a single signal that may be misinterpreted as the close association of two homologous chromosomal sites. The nuclear distribution of homologous loci, when corrected for the influence of nuclear architecture and methodological faults, was not different or was only marginally different from a random relative positioning as predicted by computer simulation. We discuss here several possibilities for the residual homologous proximity that do not invoke homology-dependent vegetative pairing, and we conclude that, in diploid budding yeast, constitutive vegetative pairing is a negligible factor for the organization of the interphase nucleus.