We identify a large coiled-coil protein, Sme4/PaMe4, that is highly conserved among the large group of Sordariales and plays central roles in two temporally and functionally distinct aspects of the fungal sexual cycle: first as a component of the meiotic synaptonemal complex (SC) and then, after disappearing and reappearing, as a component of the spindle pole body (SPB). In both cases, the protein mediates spatial juxtaposition of two major structures: linkage of homolog axes through the SC and a change in the SPB from a planar to a bent conformation. Corresponding mutants exhibit defects, respectively, in SC and SPB morphogenesis, with downstream consequences for recombination and astral-microtubule nucleation plus postmeiotic nuclear migration. Sme4 is also required for reorganization of recombination complexes in which Rad51, Mer3, and Msh4 foci relocalize from an on-axis position to a between-axis (on-SC) position concomitant with SC installation. Because involved recombinosome foci represent total recombinational interactions, these dynamics are irrespective of their designation for maturation into cross-overs or noncross-overs. The defined dual roles for Sme4 in two different structures that function at distinct phases of the sexual cycle also provide more functional links and evolutionary dynamics among the nuclear envelope, SPB, and SC.

Repair of DNA double-strand breaks (DSBs) by homologous recombination is crucial for cell proliferation and tumour suppression. However, despite its importance, the molecular intermediates of mitotic DSB repair remain undefined. The double Holliday junction (DHJ), presupposed to be the central intermediate for more than 25 years, has only been identified during meiotic recombination. Moreover, evidence has accumulated for alternative, DHJ-independent mechanisms, raising the possibility that DHJs are not formed during DSB repair in mitotically cycling cells. Here we identify intermediates of DSB repair by using a budding-yeast assay system designed to mimic physiological DSB repair. This system uses diploid cells and provides the possibility for allelic recombination either between sister chromatids or between homologues, as well as direct comparison with meiotic recombination at the same locus. In mitotically cycling cells, we detect inter-homologue joint molecule (JM) intermediates whose strand composition and size are identical to those of the canonical DHJ structures observed in meiosis. However, in contrast to meiosis, JMs between sister chromatids form in preference to those between homologues. Moreover, JMs seem to represent a minor pathway of DSB repair in mitotic cells, being detected at about tenfold lower levels (per DSB) than during meiotic recombination. Thus, although DHJs are identified as intermediates of DSB-promoted recombination in both mitotic and meiotic cells, their formation is distinctly regulated according to the specific dictates of the two cellular programs.

Meiotic chromosome pairing involves not only recognition of homology but also juxtaposition of entire chromosomes in a topologically regular way. Analysis of filamentous fungus Sordaria macrospora reveals that recombination proteins Mer3, Msh4, and Mlh1 play direct roles in all of these aspects, in advance of their known roles in recombination. Absence of Mer3 helicase results in interwoven chromosomes, thereby revealing the existence of features that specifically ensure "entanglement avoidance." Entanglements that remain at zygotene, i.e., "interlockings," require Mlh1 for resolution, likely to eliminate constraining recombinational connections. Patterns of Mer3 and Msh4 foci along aligned chromosomes show that the double-strand breaks mediating homologous alignment have spatially separated ends, one localized to each partner axis, and that pairing involves interference among developing interhomolog interactions. We propose that Mer3, Msh4, and Mlh1 execute all of these roles during pairing by modulating the state of nascent double-strand break/partner DNA contacts within axis-associated recombination complexes.

Meiotic double-strand break (DSB)-initiated recombination must occur between homologous maternal and paternal chromosomes ("homolog bias"), even though sister chromatids are present. Through physical recombination analyses, we show that sister cohesion, normally mediated by meiotic cohesin Rec8, promotes "sister bias"; that meiosis-specific axis components Red1/Mek1kinase counteract this effect, thereby satisfying an essential precondition for homolog bias; and that other components, probably recombinosome-related, directly ensure homolog partner selection. Later, Rec8 acts positively to ensure maintenance of bias. These complexities mirror opposing dictates for global sister cohesion versus local separation and differentiation of sisters at recombination sites. Our findings support DSB formation within axis-tethered recombinosomes containing both sisters and ensuing programmed sequential release of "first" and "second" DSB ends. First-end release would create a homology-searching "tentacle." Rec8 and Red1/Mek1 also independently license recombinational progression and abundantly localize to different domains. These domains could comprise complementary environments that integrate inputs from DSB repair and mitotic chromosome morphogenesis into the complete meiotic program.

Fluorescent in situ hybridization (FISH) provides a powerful tool to study the localization of DNA sequences in relationship to one another. FISH has the advantage over other methods, notably use of GFP-tagged repressor/operator arrays, that an almost unlimited number of probes can be utilized without having to make new strains for each new locus one wants to study. Also, the number of sites that can be visualized at the same time is limited only by the number of fluorophores that are available and can be distinguished by the available microscope. Described here is a method for FISH analysis and its application to analysis of chromosome pairing during meiosis in S. cerevisiae.

Dramatic chromosome motion is a characteristic of mid-prophase of meiosis that is observed across broadly divergent eukaryotic phyla. Although the specific mechanisms underlying chromosome motions vary among organisms studied to date, the outcome is similar in all cases: vigorous back-and-forth movement (as fast as approximately 1mum/sec for budding yeast), led by chromosome ends (or near-end regions), and directed by cytoskeletal components via direct association through the nuclear envelope. The exact role(s) of these movements remains unknown, although an idea gaining currency is that movement serves as a stringency factor, eliminating unwanted inter-chromosomal associations or entanglements that have arisen as part of the homolog pairing process and, potentially, unwanted associations of chromatin with the nuclear envelope. Turbulent chromosome movements observed during bipolar orientation of chromosomes for segregation could also serve similar roles during mitosis. Recent advances shed light on the contribution of protein complexes involved in the meiotic movements in chromosome dynamics during the mitotic program.

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.

It has been suggested that the structure that results when double-stranded DNA (dsDNA) is pulled from the 3'3' ends differs from that which results when it is pulled from the 5'5' ends. In this work, we demonstrate, using lambda phage dsDNA, that the overstretched states do indeed show different properties, suggesting that they correspond to different structures. For 3'3' pulling versus 5'5' pulling, the following differences are observed: (i) the forces at which half of the molecules in the ensemble have made a complete force-induced transition to single stranded DNA are 141 +/- 3 pN and 122 +/- 4 pN, respectively; (ii) the extension vs. force curve for overstretched DNA has a marked change in slope at 127 +/- 3 pN for 3'3' and 110 +/- 3 pN for 5'5'; (iii) the hysteresis (H) in the extension vs. force curves at 150 mM NaCl is 0.3 +/- 0.8 pN microm for 3'3' versus 13 +/- 8 pN for 5'5'; and (iv) 3'3' and 5'5' molecules show different changes in hysteresis due to interactions with beta-cyclodextrin, a molecule that is known to form stable host-guest complexes with rotated base pairs, and glyoxal that is known to bind stably to unpaired bases. These differences and additional findings are well-accommodated by the corresponding structures predicted on theoretical grounds.

During meiosis, DNA events of recombination occur in direct physical association with underlying chromosome axes. Meiotic cohesin Rec8 and cohesin-associated Spo76/Pds5 are prominent axis components. Two observations indicate that recombination complexes can direct the local destabilization of underlying chromosome axes. First, in the absence of Rec8, Spo76/Pds5 is lost locally at sites of late-persisting Msh4 foci, with a concomitant tendency for loosening of intersister and interhomolog connectedness at the affected sites. This loss is dependent on initiation of recombination. Second, in wild-type prophase, local separation of sister axes is seen at sites of synaptonemal complex-associated recombination nodules. Additional findings reveal that Rec8 localizes to both axis and bulk chromatin and is required for chromatin compactness. Further, Rec8 is essential for maintenance of sister cohesion, along arms and centromeres, during the pachytene-to-diplotene transition, revealing an intrinsic tendency for destabilization of sister cohesion during this period. This finding shows how the loss of sister connectedness, in arm and/or centric regions, could lead to the segregation defects that are seen in the human "maternal age effect" and how Rec8 could be a target of that effect. Finally, Rec8 plays related, but synergistic roles with Spo76/Pds5, indicating auxiliary roles for meiotic and mitotic cohesion-associated components.

The probability with which different regions of a genome come in contact with one another is a question of general interest. The current study addresses this subject for vegetatively growing diploid cells of fission yeast Schizosaccharomyces pombe by application of the Cre/loxP site-specific recombination assay. High levels of allelic interactions imply a tendency for chromosomes to be colocalized along their lengths. Significant homology-dependent pairing at telomere proximal loci and robust nonspecific clustering of centromeres appear to be the primary determinants of this feature. Preference for direct homolog-directed interactions at interstitial chromosomal regions was ambiguous, perhaps as a consequence of chromosome flexibility and the constraints and dynamic nature of the nucleus. Additional features of the data provide evidence for chromosome territories and reveal an intriguing phenomenon in which interaction frequencies are favored for nonhomologous loci that are located at corresponding relative (rather than absolute) positions within their respective chromosome arms. The latter feature, and others, can be understood as manifestations of transient, variable, and/or occasional nonspecific telomeric associations. We discuss the factors whose interplay sets the probabilities of chromosomal interactions in this organism and implications of the inferred organization for ectopic recombination.

We show that, during budding yeast meiosis, axis ensemble Hop1/Red1 and synaptonemal complex (SC) component Zip1 tend to occur in alternating strongly staining domains. The widely conserved AAA+-ATPase Pch2 mediates this pattern, likely by means of direct intervention along axes. Pch2 also coordinately promotes timely progression of cross-over (CO) and noncross-over (NCO) recombination. Oppositely, in a checkpoint-triggering aberrant situation (zip1Delta), Pch2 mediates robust arrest of stalled recombination complexes, likely via nucleolar localization. We suggest that, during WT meiosis, Pch2 promotes progression of SC-associated CO and NCO recombination complexes at a regulated early-midpachytene transition that is rate-limiting for later events; in contrast, during defective meiosis, Pch2 ensures that aberrant recombination complexes fail to progress so that intermediates can be harmlessly repaired during eventual return to growth. Positive vs. negative roles of Pch2 in the two situations are analogous to positive vs. negative roles of Mec1/ATR, suggesting that Pch2 might mediate Mec1/ATR activity. We further propose that regulatory surveillance of normal and abnormal interchromosomal interactions in mitotic and meiotic cells may involve "structure-dependent interchromosomal interaction" (SDIX) checkpoints.

Chromosome movements are a general feature of mid-prophase of meiosis. In budding yeast, meiotic chromosomes exhibit dynamic movements, led by nuclear envelope (NE)-associated telomeres, throughout the zygotene and pachytene stages. Zygotene motion underlies the global tendency for colocalization of NE-associated chromosome ends in a "bouquet." In this study, we identify Csm4 as a new molecular participant in these processes and show that, unlike the two previously identified components, Ndj1 and Mps3, Csm4 is not required for meiosis-specific telomere/NE association. Instead, it acts to couple telomere/NE ensembles to a force generation mechanism. Mutants lacking Csm4 and/or Ndj1 display the following closely related phenotypes: (i) elevated crossover (CO) frequencies and decreased CO interference without abrogation of normal pathways; (ii) delayed progression of recombination, and recombination-coupled chromosome morphogenesis, with resulting delays in the MI division; and (iii) nondisjunction of homologs at the MI division for some reason other than absence of (the obligatory) CO(s). The recombination effects are discussed in the context of a model where the underlying defect is chromosome movement, the absence of which results in persistence of inappropriate chromosome relationships that, in turn, results in the observed mutant phenotypes.

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.

SUMMARY: In Escherichia coli, the chemotaxis receptor protein Tsr localizes abundantly to cell poles. The current study, utilizing a Tsr-GFP fusion protein and time-lapse fluorescence microscopy of individual cell lineages, demonstrates that Tsr accumulates approximately linearly with time at the cell poles and that, in consequence, more Tsr is present at the old pole of each cell than at its newborn pole. The rate of pole-localized Tsr accumulation is large enough that old and new poles can always be reliably distinguished, even for cells whose old poles have had only one generation to accumulate signal. Correspondingly, Tsr-GFP can be reliably used to assign new and old poles to any cell without use of information regarding pole heritage, thus providing a useful tool to analyse cells whose prior history is not available. The absolute level of Tsr-GFP at the old pole of a cell also provides a rough estimate of pole (and thus cell) age.

Meiotic recombination-related DNA synthesis (MRDS) was analyzed in Saccharomyces cerevisiae by specifically timed incorporation of thymidine analogs into chromosomes. Lengths and positions of incorporation tracts were determined relative to a known recombination hot spot along DNA, as was the timing and localization of incorporation relative to forming and formed synaptonemal complex in spread chromosomes. Distinct patterns could be specifically associated with the majority cross-over and non-cross-over recombination processes. The results obtained provide direct evidence for key aspects of current consensus recombination models, provide information regarding temporal and spatial relationships between non-cross-over formation and the synaptonemal complex, and raise the possibility that removal of RecA homolog Rad51 plays a key role in regulating onset of MRDS. Finally, classical observations on MRDS in Drosophila, mouse, and lily are readily mapped onto the findings presented here, providing further evidence for a broadly conserved meiotic recombination process.

Meiotic recombination proceeds in biochemical complexes that are physically associated with underlying chromosome structural axes. In this study, we discuss the organizational basis for these axes, the timing and nature of recombinosome/axis organization with respect to the prophase program of DNA and to structural changes, and the possible significance of axis organization. Furthermore, we discuss implications and extensions of our recently proposed mechanical model for chiasma formation. Finally, we give a broader consideration to past and present models for the role of the synaptonemal complex.

Zip2 and Zip3 are meiosis-specific proteins that, in collaboration with several partners, act at the sites of crossover-designated, axis-associated recombinational interactions to mediate crossover/chiasma formation. Here, Spo22 (also called Zip4) is identified as a probable functional collaborator of Zip2/3. The molecular roles of Zip2, Zip3, and Spo22/Zip4 are unknown. All three proteins are part of a small evolutionary cohort comprising similar homologs in four related yeasts. Zip3 is shown to contain a RING finger whose structural features most closely match those of known ubiquitin E3s. Further, Zip3 exhibits major domainal homologies to Rad18, a known DNA-binding ubiquitin E3. Also described is an approach to the identification and mapping of repeated protein sequence motifs, Alignment Based Repeat Annotation (ABRA), that we have developed. When ABRA is applied to Zip2 and Spo22/Zip4, they emerge as a 14-blade WD40-like repeat protein and a 22-unit tetratricopeptide repeat protein, respectively. WD40 repeats of Cdc20, Cdh1, and Cdc16 and tetratricopeptide repeats of Cdc16, Cdc23, and Cdc27, all components of the anaphase-promoting complex, are also analyzed. These and other findings suggest that Zip2, Zip3, and Zip4 act together to mediate a process that involves Zip3-mediated ubiquitin labeling, potentially as a unique type of ubiquitin-conjugating complex.

Chromosome and replisome dynamics were examined in synchronized E. coli cells undergoing a eukaryotic-like cell cycle. Sister chromosomes remain tightly colocalized for much of S phase and then separate, in a single coordinate transition. Origin and terminus regions behave differently, as functionally independent domains. During separation, sister loci move far apart and the nucleoid becomes bilobed. Origins and terminus regions also move. We infer that sisters are initially linked and that loss of cohesion triggers global chromosome reorganization. This reorganization creates the 2-fold symmetric, ter-in/ori-out conformation which, for E. coli, comprises sister segregation. Analogies with eukaryotic prometaphase suggest that this could be a primordial segregation mechanism to which microtubule-based processes were later added. We see no long-lived replication "factory"; replication initiation timing does not covary with cell mass, and we identify changes in nucleoid position and state that are tightly linked to cell division. We propose that cell division licenses the next round of replication initiation via these changes.

We describe a new method for synchronizing bacterial cells. Cells that have transiently expressed an inducible mutant 'sticky' flagellin are adhered to a volume of glass beads suspended in a chromatography column though which growth medium is pumped. Following repression of flagellin synthesis, newborn cells are eluted from the column in large quantities exceeding that of current baby machine techniques by approximately 10-fold. Eluted cultures of 'baby cells' are highly synchronous as determined by analysis of DNA replication, cell division and other events, over time after elution from the column. We also show that use of 'minutes after elution' as a time metric permits much greater temporal resolution among sequential chromosomal events than the commonly used metric of cell size (length). The former approach reveals the existence of transient intermediate stages that are missed by the latter approach. This finding has two important implications. First, at a practical level, the baby cell column is a particularly powerful method for temporal analysis. Second, at a conceptual level, replication-related events are more tightly linked to cell birth (i.e. cell division) than to absolute cell mass.