Meiotic silencing by unpaired DNA (MSUD) is a process that detects unpaired regions between homologous chromosomes and silences them for the duration of sexual development. While the phenomenon of MSUD is well recognized, the process that detects unpaired DNA is poorly understood. In this report, we provide two lines of evidence linking unpaired DNA detection to a physical search for DNA homology. First, we have found that a putative SNF2-family protein (SAD-6) is required for efficient MSUD in Neurospora crassa. SAD-6 is closely related to Rad54, a protein known to facilitate key steps in the repair of double-strand breaks by homologous recombination. Second, we have successfully masked unpaired DNA by placing identical transgenes at slightly different locations on homologous chromosomes. This masking falls apart when the distance between the transgenes is increased. We propose a model where unpaired DNA detection during MSUD is achieved through a spatially constrained search for DNA homology. The identity of SAD-6 as a Rad54 paralog suggests that this process may be similar to the searching mechanism used during homologous recombination. MEIOSIS is fundamental to sexual reproduction. During meiosis, chromosomes are replicated, aligned, recombined, and segregated to nuclei that will develop into gametes. Two of these key processes, alignment and recombination, likely require a search for DNA homology between chromosomes (Barzel and Kupiec 2008;Moore and Shaw 2009). Such homology searching is necessary because sexual organisms inherit a copy of each chromosome from each of its parents. These chromosomes, referred to as homologs, must somehow find each other so that alignment, recombination, and segregation can occur.Although recent research has improved our understanding of homology search mechanisms (Forget and Kowalczykowski 2012;Renkawitz et al. 2013), there are many questions that remain unanswered. The filamentous fungus Neurospora crassa may be useful for investigating the unknowns of homology searching because it possesses a genetically tractable phenomenon called meiotic silencing by unpaired DNA (MSUD) (Aramayo and Selker 2013;Billmyre et al. 2013). MSUD scans pairs of homologs for segments of DNA that are not accurately paired between them. If improper pairing (i.e., unpairing) is identified, the offending sequences are silenced for the duration of sexual development. For example, if a hypothetical gene called "gene A" is on the left arm of one chromosome but on the right arm of its homolog, it will be silenced. The same holds true if gene A has been lost from one of the homologs.A functional MSUD response can be easily detected with alleles that affect ascospore (sexual spore) color or shape. Indeed, MSUD was discovered during studies of ascospore maturation-1 (asm-1), a gene required for the production of pigmented (black) ascospores . A cross between an asm-1 + strain and an asm-1 D strain produces mostly unpigmented (white) ascospores. This is because MSUD silences the unpaired asm-1 + all...

Neurospora fungi harbor a group of meiotic drive elements known as Spore killers (Sk). Spore killer-2 (Sk-2) and Spore killer-3 (Sk-3) are two Sk elements that map to a region of suppressed recombination. Although this recombination block is limited to crosses between Sk and Sk-sensitive (Sk S ) strains, its existence has hindered Sk characterization. Here we report the circumvention of this obstacle by combining a classical genetic screen with next-generation sequencing technology and three-point crossing assays. This approach has allowed us to identify a novel locus called rfk-1, mutation of which disrupts spore killing by Sk-2. We have mapped rfk-1 to a 45-kb region near the right border of the Sk-2 element, a location that also harbors an 11-kb insertion (Sk-2 INS1 ) and part of a .220-kb inversion (Sk-2 INV1 ). These are the first two chromosome rearrangements to be formally identified in a Neurospora Sk element, providing evidence that they are at least partially responsible for Sk-based recombination suppression. Additionally, the proximity of these chromosome rearrangements to rfk-1 (a critical component of the spore-killing mechanism) suggests that they have played a key role in the evolution of meiotic drive in Neurospora.G ENOMES typically consist of large groups of cooperating genes that encode information needed to maintain and reproduce themselves as well as their cellular environments. However, individual genes within genomes do not always cooperate (Dawkins 2006;Burt and Trivers 2008). For example, ranGAP in fruit flies and tcd1 in mice are selfish genes that collude with others to bias meiotic transmission ratios to their advantage (Kusano et al. 2003;Lyon 2003). While ranGAP works within a gene complex known as Segregation Distorter, tcd1 works within another called the t haplotype. Because these selfish gene complexes "drive" through meiosis, they are referred to as meiotic drive elements. Such elements are found throughout sexual organisms, with other well-known examples being X d in stalkeyed flies, Ab10 in maize, and the Spore killers in various fungi (Rhoades 1942;Raju 1994;Presgraves et al. 1997;Burt and Trivers 2008).Neurospora fungi spend most of their life cycle in a vegetative haploid phase. However, specific environmental cues (e.g., nitrogen limitation) can trigger initiation of the sexual cycle, which includes a brief diploid phase that allows meiotic drive to function in this group of fungi. Sexual reproduction in Neurospora begins when an asexual spore (conidium) from a male parent donates a nucleus to an immature fruiting body (protoperithecium) of a female parent. Pairs of nuclei, one from each parent, fuse within the meiotic cells (asci) of the fertilized fruiting body (perithecium) to form a diploid nucleus that passes through the standard stages of meiosis (Raju 1980). The four haploid meiotic products then undergo a postmeiotic mitosis to produce eight nuclei, each of which is incorporated into an American football-shaped ascospore (Raju 1980). Ascospores darken as...

BIOLOGICAL BACKGROUND 1.1: Biological Background This section is intended to give a brief description of the biological processes that are relevant to this project. 1.1.1: Meiosis Meiosis is a specific type of cell division used for the production of sex cells (spores in the case of fungi) in eukaryotes. The steps involved in meiotic cell division are similar to those for other somatic cells, which divide through a well-known process called mitosis. Meiosis, shown in Figure 1, begins with a duplication of chromosomes (Figure 1a). Next, each chromosome pairs with its homolog (Figure 1b). This pairing allows for the exchange of genetic material, which contributes to genetic variation, through a process called crossing over. Homologous chromosomes are moved to opposite poles of the cell and the original cell is then split into two daughter cells (Figure 1c). This process repeats again, this time without initial duplication of the chromosomes (Figure 1d). This results in each of the two daughter cells dividing to produce four cells with half the genetic information (Figure 1e).