Magnetic force micropiston: an integrated force/microfluidic device for the application of compressive forces in a confined environment.

Cellular biology takes place inside confining spaces. For example, bacteria grow in crevices, red blood cells squeeze through capillaries, and chromosomes replicate inside the nucleus. Frequently, the extent of this confinement varies. Bacteria grow longer and divide, red blood cells move through smaller and smaller passages as they travel to capillary beds, and replication doubles the amount of DNA inside the nucleus. This increase in confinement, either due to a decrease in the available space or an increase in the amount of material contained in a constant volume, has the potential to squeeze and stress objects in ways that may lead to changes in morphology, dynamics, and ultimately biological function. Here, we describe a device developed to probe the interplay between confinement and the mechanical properties of cells and cellular structures, and forces that arise due to changes in a structure's state. In this system, the manipulation of a magnetic bead exerts a compressive force upon a target contained in the confining space of a microfluidic channel. This magnetic force microfluidic piston is constructed in such a way that we can measure (a) target compliance and changes in compliance as induced by changes in buffer, extract, or biochemical composition, (b) target expansion force generated by changes in the same parameters, and (c) the effects of compression stress on a target's structure and function. Beyond these issues, our system has general applicability to a variety of questions requiring the combination of mechanical forces, confinement, and optical imaging.

Single molecule detection of direct, homologous, DNA/DNA pairing.

Using a parallel single molecule magnetic tweezers assay we demonstrate homologous pairing of two double-stranded (ds) DNA molecules in the absence of proteins, divalent metal ions, crowding agents, or free DNA ends. Pairing is accurate and rapid under physiological conditions of temperature and monovalent salt, even at DNA molecule concentrations orders of magnitude below those found in vivo, and in the presence of a large excess of nonspecific competitor DNA. Crowding agents further increase the reaction rate. Pairing is readily detected between regions of homology of 5 kb or more. Detected pairs are stable against thermal forces and shear forces up to 10 pN. These results strongly suggest that direct recognition of homology between chemically intact B-DNA molecules should be possible in vivo. The robustness of the observed signal raises the possibility that pairing might even be the "default" option, limited to desired situations by specific features. Protein-independent homologous pairing of intact dsDNA has been predicted theoretically, but further studies are needed to determine whether existing theories fit sequence length, temperature, and salt dependencies described here.

Meiotic chromosomes move by linkage to dynamic actin cables with transduction of force through the nuclear envelope.

Chromosome movement is prominent during meiosis. Here, using a combination of in vitro and in vivo approaches, we elucidate the basis for dynamic mid-prophase telomere-led chromosome motion in budding yeast. Diverse findings reveal a process in which, at the pachytene stage, individual telomere/nuclear envelope (NE) ensembles attach passively to, and then move in concert with, nucleus-hugging actin cables that are continuous with the global cytoskeletal actin network. Other chromosomes move in concert with lead chromosome(s). The same process, in modulated form, explains the zygotene "bouquet" configuration in which, immediately preceding pachytene, chromosome ends colocalize dynamically in a restricted region of the NE. Mechanical properties of the system and biological roles of mid-prophase movement for meiosis, including recombination, are discussed.

The single-end invasion: an asymmetric intermediate at the double-strand break to double-holliday junction transition of meiotic recombination.

We identify a novel meiotic recombination intermediate, the single-end invasion (SEI), which occurs during the transition from double-strand breaks (DSBs) to double-Holliday junction (dHJs). SEIs are products of strand exchange between one DSB end and its homolog. The structural asymmetry of SEIs indicates that the two ends of a DSB interact with the homolog in temporal succession, via structurally (and thus biochemically) distinct processes. SEIs arise surprisingly late in prophase, concomitant with synaptonemal complex (SC) formation. These and other data imply that SEIs are preceded by nascent DSB-partner intermediates, which then undergo selective differentiation into crossover and noncrossover types, with SC formation and strand exchange as downstream consequences. Late occurrence of strand exchange provides opportunity to reverse recombinational fate even after homologs are coaligned and/or synapsed. This feature can explain crossover suppression between homeologous and structurally heterozygous chromosomes.

Tn10 transpososome assembly involves a folded intermediate that must be unfolded for target capture and strand transfer.

Tn10 transposition, like all transposition reactions examined thus far, involves assembly of a stable protein-DNA transpososome, containing a pair of transposon ends, within which all chemical events occur. We report here that stable Tn10 pre-cleavage transpososomes occur in two conformations: a folded form which contains the DNA-bending factor IHF and an unfolded form which lacks IHF. Functional analysis shows that both forms undergo double strand cleavage at the transposon ends but that only the unfolded form is competent for target capture (and thus for strand transfer to target DNA). Additional studies reveal that formation of any type of stable transpososome, folded or unfolded, requires not only IHF but also non-specific transposase-DNA contacts immediately internal to the IHF-binding site, implying the occurrence of a topo- logically closed loop at the transposon end. Overall, transpososome assembly must proceed via a folded intermediate which, however, must be unfolded in order for intermolecular transposition to occur. These and other results support key features of a recently proposed model for transpososome assembly and morphogenesis.

Progression of meiotic DNA replication is modulated by interchromosomal interaction proteins, negatively by Spo11p and positively by Rec8p.

Spo11p is a key mediator of interhomolog interactions during meiosis. Deletion of the SPO11 gene decreases the length of S phase by approximately 25%. Rec8p is a key coordinator of meiotic interhomolog and intersister interactions. Deletion of the REC8 gene increases S-phase length, by approximately 10% in wild-type and approximately 30% in a spo11Delta background. Thus, the progression of DNA replication is modulated by interchromosomal interaction proteins. The spo11-Y135F DSB (double strand break) catalysis-defective mutant is normal for S-phase modulation and DSB-independent homolog pairing but is defective for later events, formation of DSBs, and synaptonemal complexes. Thus, earlier and later functions of Spo11 are defined. We propose that meiotic S-phase progression is linked directly to development of specific chromosomal features required for meiotic interhomolog interactions and that this feedback process is built upon a more fundamental mechanism, common to all cell types, by which S-phase progression is coupled to development of nascent intersister connections and/or related aspects of chromosome morphogenesis. Roles for Rec8 and/or Spo11 in progression through other stages of meiosis are also revealed.

Functional specificity of MutL homologs in yeast: evidence for three Mlh1-based heterocomplexes with distinct roles during meiosis in recombination and mismatch correction.

The yeast genome encodes four proteins (Pms1 and Mlh1-3) homologous to the bacterial mismatch repair component, MutL. Using two hybrid-interaction and coimmunoprecipitation studies, we show that these proteins can form only three types of complexes in vivo. Mlh1 is the common component of all three complexes, interacting with Pms1, Mlh2, and Mlh3, presumptively as heterodimers. The phenotypes of single deletion mutants reveal distinct functions for the three heterodimers during meiosis: in a pms1 mutant, frequent postmeiotic segregation indicates a defect in the correction of heteroduplex DNA, whereas the frequency of crossing-over is normal. Conversely, crossing-over in the mlh3 mutant is reduced to approximately 70% of wild-type levels but correction of heteroduplex is normal. In a mlh2 mutant, crossing-over is normal and postmeiotic segregation is not observed but non-Mendelian segregation is elevated and altered with respect to parity. Finally, to a first approximation, the mlh1 mutant represents the combined single mutant phenotypes. Taken together, these data imply modulation of a basic Mlh1 function via combination with the three other MutL homologs and suggest specifically that Mlh1 combines with Mlh3 to promote meiotic crossing-over.

Cohesins bind to preferential sites along yeast chromosome III, with differential regulation along arms versus the centric region.

Sister chromatid cohesion is mediated by evolutionary conserved chromosomal proteins, termed "cohesins." Using an extension of chromatin immunoprecipitation, we have analyzed the distribution of cohesins Mcd1/ Sccl and Smc1 along yeast chromosome III. Both proteins occur preferentially at the same approximately 23 positions. Sites in a approximately 50 kb region around the centromere give especially intense signals. Prominent centric region binding appears to emerge from a more even distribution, probably by differential loss of cohesins along the chromosome arms. Cohesin binding peaks correspond closely to peaks of high local AT composition, a base composition periodicity of approximately 15 kb that is distinct from the approximately 50 kb periodicity of base composition isochores, consistent with axis association of cohesins. The methodology described can be used to analyze the distribution of any DNA-binding protein and, via microchips, along entire genomes.

Somatic pairing of homologs in budding yeast: existence and modulation.

FISH analysis of well-spread chromosomes reveals that homologs are paired in vegetatively growing budding yeast diploid cells, via multiple interstitial interactions, and independent of recA homologs and mating type heterozygosity. Pairing is present during G1 and G2, and in cells arrested at G1 by mating pheromone, but is disrupted during S phase. Thus, somatic pairing is qualitatively analogous to premeiotic and early meiotic pairing. S-phase pairing disruption occurs by a complex intranuclear program involving regional, nucleus-wide, and temporal determinants. Pairing is also disrupted in two G2-arrest conditions (cdc13ts and nocodazole). Together these findings suggest that cell cycle signals may provoke pairing disruption by modulating underlying chromosome and/or chromatin structure. Whether the cell chooses to disrupt pairing contacts or not (e.g., S phase and G2 arrest, but not G1 arrest or normal G1 or G2), could be dictated by functional considerations involving homolog/sister discrimination.

Collisions between yeast chromosomal loci in vivo are governed by three layers of organization.

The relative probabilities that different pairs of chromosomal loci will collide with one another in vegetatively growing diploid yeast cells have been assessed using a genetic assay for Cre/loxP site-specific recombination. Recombination rates have been determined for 18 different pairs of loxP sites representing diverse pairs of positions within the genome. Overall, relative collision probabilities vary over an eightfold range. Within this range, a hierarchy comprising three levels of organization can be discerned. First, collisions between loci on nonhomologous chromosomes are governed by nonspecific centromere clustering. Second, a sequence is closer to allelic or nearby sequences on its homolog than to sequences on nonhomologous chromosomes, an effect most simply attributed to homolog pairing. Third, a sequence can be closer to other sequences nearby on the same chromosome than to sequences on other chromosomes. These findings provide a framework for assessing the role of chromosome disposition in cellular processes such as DNA repair and gene expression. Also the possibility is raised that genome-wide coalignment of homologs is not the fundamental raison d'etre of the somatic pairing process. We suggest instead that pairing may exist to promote juxtaposition of homologous regions within irregular genome complements.

GCN5-dependent histone H3 acetylation and RPD3-dependent histone H4 deacetylation have distinct, opposing effects on IME2 transcription, during meiosis and during vegetative growth, in budding yeast.

Diploid yeast undergo meiosis under certain conditions of nutrient limitation, which trigger a transcriptional cascade involving two key regulatory genes. IME1 is a positive activator of IME2, which activates downstream genes. We report that Gcn5, a histone H3 acetylase, plays a central role in initiation of meiosis via effects on IME2 expression. An allele, gcn5-21, was isolated as a mutant defective in spore formation. gcn5-21 fails to carry out meiotic DNA replication, recombination, or meiotic divisions. This mutant also fails to induce IME2 transcription; IME1 transcription, however, is essentially normal. Further investigation shows that during wild-type meiosis the IME2 promoter undergoes an increase in the level of bound acetylated histone H3. This increase is contemporaneous with meiotic induction of IME2 transcription and is absent in gcn5-21. In contrast, the RPD3 gene, which encodes a histone H4 deacetylase and is known to be required for repression of basal IME2 transcription in growing yeast cells, is not involved in induction of IME2 transcription or IME2 histone acetlyation during meiosis. These and other results suggest that Gcn5 and Rpd3 play distinct roles, modulating transcription initiation in opposite directions under two different cellular conditions. These roles are implemented via opposing effects of the two gene products on acetylation of two different histones. Finally, we find that gcn5 and rpd3 single mutants are not defective in meiosis if acetate is absent and respiration is promoted by a metabolically unrelated carbon source. Perhaps intracellular acetate levels regulate meiosis by controlling histone acetylation patterns.

Subtype-selective antagonism of N-methyl-D-aspartate receptors by felbamate: insights into the mechanism of action.

Felbamate is an anticonvulsant used in the treatment of seizures associated with Lennox-Gastaut syndrome and complex partial seizures that are refractory to other medications. Its unique clinical profile is thought to be due to an interaction with N-methyl-D-aspartate (NMDA) receptors, resulting in decreased excitatory amino acid neurotransmission. To further characterize the interaction between felbamate and NMDA receptors, recombinant receptors expressed in Xenopus oocytes were used to investigate the subtype specificity and mechanism of action. Felbamate reduced NMDA- and glycine-induced currents most effectively at NMDA receptors composed of NR1 and NR2B subunits (IC50 = 0.93 mM), followed by NR1-2C (2.02 mM) and NR1-2A (8.56 mM) receptors. The NR1-2B-selective interaction was noncompetitive with respect to the coagonists NMDA and glycine and was not dependent on voltage. Felbamate enhanced the affinity of the NR1-2B receptor for the agonist NMDA by 3.5-fold, suggesting a similarity in mechanism to other noncompetitive antagonists such as ifenprodil. However, a point mutation at position 201 (E201R) of the epsilon2 (mouse NR2B) subunit that affects receptor sensitivity to ifenprodil, haloperidol, and protons reduced the affinity of NR1-epsilon2 receptors for felbamate by only 2-fold. Furthermore, pH had no effect on the affinity of NR1-2B receptors for felbamate. We suggest that felbamate interacts with a unique site on the NR2B subunit (or one formed by NR1 plus NR2B) that interacts allosterically with the NMDA/glutamate binding site. These results suggest that the unique clinical profile of felbamate is due in part to an interaction with the NR1-2B subtype of NMDA receptor.

Meiotic chromosomes: integrating structure and function.

Meiotic chromosomes have been studied for many years, in part because of the fundamental life processes they represent, but also because meiosis involves the formation of homolog pairs, a feature which greatly facilitates the study of chromosome behavior. The complex events involved in homolog juxtaposition necessitate prolongation of prophase, thus permitting resolution of events that are temporally compressed in the mitotic cycle. Furthermore, once homologs are paired, the chromosomes are connected by a specific structure: the synaptonemal complex. Finally, interaction of homologs includes recombination at the DNA level, which is intimately linked to structural features of the chromosomes. In consequence, recombination-related events report on diverse aspects of chromosome morphogenesis, notably relationships between sisters, development of axial structure, and variations in chromatin status. The current article reviews recent information on these topics in an historical context. This juxtaposition has suggested new relationships between structure and function. Additional issues were addressed in a previous chapter (551).

Tn10 transposition via a DNA hairpin intermediate.

We present evidence that excision of the nonreplicative transposon Tn10 involves three distinct chemical steps, first-strand nicking, hairpin formation, and hairpin resolution. This three-step mechanism makes it possible for a single protein-active site to cleave two DNA strands of opposite polarity, as appears to be the case in this reaction. We infer the existence of alternating bifunctionality within the active site with suitable modulation of substrate components between steps. DNA double-strand breaks are also made by a "hairpin mechanism" in V(D)J recombination, possibly reflecting the same basic constraints faced in the Tn10 system. Similarities in the basic chemical steps in Tn10 transposition and V(D)J recombination suggest that the V(D)J mechanism may have evolved from a bacterial transposition system.

Effects of purified SeqA protein on oriC-dependent DNA replication in vitro.

In vivo studies suggest that the Escherichia coli SeqA protein modulates replication initiation in two ways: by delaying initiation and by sequestering newly replicated origins from undergoing re-replication. As a first approach towards understanding the biochemical bases for these effects, we have examined the effects of purified SeqA protein on replication reactions performed in vitro on an oriC plasmid. Our results demonstrate that SeqA directly affects the biochemical events occurring at oriC. First, SeqA inhibits formation of the pre-priming complex. Secondly, SeqA can inhibit replication from an established pre-priming complex, without disrupting the complex. Thirdly, SeqA alters the dependence of the replication system on DnaA protein concentration, stimulating replication at low concentrations of DnaA. Our data suggest that SeqA participates in the assembly of initiation-competent complexes at oriC and, at a later stage, influences the behaviour of these complexes.

IHF modulation of Tn10 transposition: sensory transduction of supercoiling status via a proposed protein/DNA molecular spring.

Architectural protein IHF modulates Tn10 transposition in vitro. IHF stimulates transposon excision. Also, separately, IHF forces transposon end/target DNA interactions into a constrained pathway, "channeling," that yields only unknotted intratransposon inversion circles. Negative supercoiling influences both effects, differently. We infer that IHF is an architectural catalyst: it promotes initial transpososome assembly and is then ejected from the transpososome. IHF then rebinds, altering transpososome conformation to promote channeling. We also infer that the developing transpososome is a molecular spring: DNA provides basic elasticity; a conformational change in transposase provides force; and IHF and/or supercoiling provide conformational inputs. In vivo, IHF is a sensory transducer of chromosomal supercoiling status: with supercoiling absent, IHF is "supercoiling relief factor"; with supercoiling present, stimulation and channeling comprise a homeostatic pair such that modest changes in chromosome condition strongly influence transpositional outcome.

The leptotene-zygotene transition of meiosis.

The leptotene/zygotene transition of meiosis, as defined by classical cytological studies, is the period when homologous chromosomes, already being discernible individualized entities, begin to be close together or touching over portions of their lengths. This period also includes the bouquet stage: Chromosome ends, which have already become integral components of the inner nuclear membrane, move into a polarized configuration, along with other nuclear envelope components. Chromosome movements, active or passive, also occur. The detailed nature of interhomologue interactions during this period, with special emphasis on the involvement of chromosome ends, and the overall role for meiosis and recombination of chromosome movement and, especially, the bouquet stage are discussed.

Interhomolog bias during meiotic recombination: meiotic functions promote a highly differentiated interhomolog-only pathway.

Meiotic recombination occurs preferentially between homologous nonsister chromatids rather than between sisters, opposite to the bias of mitotic recombinational repair. We have examined formation of joint molecule recombination intermediates (JMs) between homologs and between sisters in yeast strains lacking the meiotic chromosomal protein Red1, the meiotic recA homolog Dmc1, and/or mitotic recA homolog(s), Rad51, Rad55, and Rad57. Mutant phenotypes imply that most meiotic recombination occurs via an interhomolog-only pathway along which interhomolog bias is established early, prior to or during double strand break (DSB) formation, and then enforced, just at the time when DSBs initiate JM formation. A parallel, less differentiated pathway yields intersister and, probably, a few interhomolog events. Coordinate action of mitotic recA homologs as one functional unit, two functions of RED1, and an interhomolog interaction function of DMC1 are also revealed.

A general method for identifying recessive diploid-specific mutations in Saccharomyces cerevisiae, its application to the isolation of mutants blocked at intermediate stages of meiotic prophase and characterization of a new gene SAE2.

We describe a general new approach for identifying recessive mutations that affect diploid strains of yeast Saccharomyces cerevisiae and the application of this method to the identification of mutations that confer an intermediate block in meiotic prophase chromosome metabolism. The method uses a temperature-sensitive conjugation mutation ste7-1 in combination with homothallism. The mutations of interest confer a defect in spore formation that is dependent upon a gene required for initiation of meiotic recombination and development of meiosis-specific chromosome structure (SPO11). Identified in this screen were null mutations of the DMC1 gene, nonnull mutations of RAD50 (rad50S), and mutations in three new genes designed SAE1, SAE2 and SAE3 (Sporulation in the Absence of Spo Eleven). Molecular characterization of the SAE2 gene and characterization of meiotic and mitotic phenotypes of sae2 mutants are also presented. The phenotypes conferred by a sae2 null mutation are virtually indistinguishable from those conferred by the previously identified nonnull mutations of RAD50 (rad50S). Most notably, both mutations confer only weak sensitivity to the radiomimetic agent methyl methane sulfonate (MMS) but completely block resection and turnover of meiosis-specific double-strand breaks. These observations provide further evidence that this constellation of phenotypes identifies a specific molecular function.