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Nancy Kleckner

Publications and source records attributed to Nancy Kleckner.

15 recordsLinked to original sources
PubMed2006

Chiasma formation: chromatin/axis interplay and the role(s) of the synaptonemal complex.

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.

Chromatin↗
PubMed2005

Bioinformatic analyses implicate the collaborating meiotic crossover/chiasma proteins Zip2, Zip3, and Spo22/Zip4 in ubiquitin labeling.

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.

Amino Acid Sequence↗
PubMed2005

Chromosome and replisome dynamics in E. coli: loss of sister cohesion triggers global chromosome movement and mediates chromosome segregation.

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.

Chromosome Mapping↗
PubMed2005

The Escherichia coli baby cell column: a novel cell synchronization method provides new insight into the bacterial cell cycle.

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.

Adhesins, Bacterial↗
PubMed2004

A mechanical basis for chromosome function.

We propose that chromosome function is governed by internal mechanical forces generated by programmed tendencies for expansion of the DNA/chromatin fiber against constraining features.

Animals↗
PubMed2004

Crossover/noncrossover differentiation, synaptonemal complex formation, and regulatory surveillance at the leptotene/zygotene transition of meiosis.

Yeast mutants lacking meiotic proteins Zip1, Zip2, Zip3, Mer3, and/or Msh5 (ZMMs) were analyzed for recombination, synaptonemal complex (SC), and meiotic progression. At 33 degrees C, recombination-initiating double-strand breaks (DSBs) and noncrossover products (NCRs) form normally while formation of single-end invasion strand exchange intermediates (SEIs), double Holliday junctions, crossover products (CRs), and SC are coordinately defective. Thus, during wild-type meiosis, recombinational interactions are differentiated into CR and NCR types very early, prior to onset of stable strand exchange and independent of SC. By implication, crossover interference does not require SC formation. We suggest that SC formation may require interference. Subsequently, CR-designated DSBs undergo a tightly coupled, ZMM-promoted transition that yields SEI-containing recombination complexes embedded in patches of SC. zmm mutant phenotypes differ strikingly at 33 degrees C and 23 degrees C, implicating higher temperature as a positive effector of recombination and identifying a checkpoint that monitors local CR-specific events, not SC formation, at late leptotene.

Chromosome Segregation↗
PubMed2003

Localization and roles of Ski8p protein in Sordaria meiosis and delineation of three mechanistically distinct steps of meiotic homolog juxtaposition.

Ski8p is implicated in degradation of non-poly(A) and double-stranded RNA, and in meiotic DNA recombination. We have identified the Sordaria macrospora SKI8 gene. Ski8p is cytoplasmically localized in all vegetative and sexual cycle cells, and is nuclear localized, specifically in early-mid-meiotic prophase, in temporal correlation with Spo11p, the meiotic double-strand break (DSB) transesterase. Localizations of Ski8p and Spo11p are mutually interdependent. ski8 mutants exhibit defects in vegetative growth, entry into the sexual program, and sporulation. Diverse meiotic defects, also seen in spo11 mutants, are diagnostic of DSB absence, and they are restored by exogenous DSBs. These results suggest that Ski8p promotes meiotic DSB formation by acting directly within meiotic prophase chromosomes. Mutant phenotypes also divide meiotic homolog juxtaposition into three successive, mechanistically distinct steps; recognition, presynaptic alignment, and synapsis, which are distinguished by their differential dependence on DSBs.

Chromosome Mapping↗
PubMed2003

Meiotic double-strand breaks at the interface of chromosome movement, chromosome remodeling, and reductional division.

Chromosomal processes related to formation and function of meiotic chiasmata have been analyzed in Sordaria macrospora. Double-strand breaks (DSBs), programmed or gamma-rays-induced, are found to promote four major events beyond recombination and accompanying synaptonemal complex formation: (1) juxtaposition of homologs from long-distance interactions to close presynaptic coalignment at midleptotene; (2) structural destabilization of chromosomes at leptotene/zygotene, including sister axis separation and fracturing, as revealed in a mutant altered in the conserved, axis-associated cohesin-related protein Spo76/Pds5p; (3) exit from the bouquet stage, with accompanying global chromosome movements, at zygotene/pachytene (bouquet stage exit is further found to be a cell-wide regulatory transition and DSB transesterase Spo11p is suggested to have a new noncatalytic role in this transition); (4) normal occurrence of both meiotic divisions, including normal sister separation. Functional interactions between DSBs and the spo76-1 mutation suggest that Spo76/Pds5p opposes local destabilization of axes at developing chiasma sites and raise the possibility of a regulatory mechanism that directly monitors the presence of chiasmata at metaphase I. Local chromosome remodeling at DSB sites appears to trigger an entire cascade of chromosome movements, morphogenetic changes, and regulatory effects that are superimposed upon a foundation of DSB-independent processes.

Chromatin↗
PubMed2003

Intra-G1 arrest in response to UV irradiation in fission yeast.

G1 is a crucial phase of cell growth because the decision to begin another mitotic cycle is made during this period. Occurrence of DNA damage in G1 poses a particular challenge, because replication of damaged DNA can be deleterious and because no sister chromatid is present to provide a template for recombinational repair. We therefore have studied the response of Schizosaccharomyces pombe cells to UV irradiation in early G1 phase. We find that irradiation results in delayed progression through G1, as manifested most critically in the delayed formation of the pre-replication complex. This delay does not have the molecular hallmarks of known checkpoint responses: it is independent of the checkpoint proteins Rad3, Cds1, and Chk1 and does not elicit inhibitory phosphorylation of Cdc2. Irradiated cells eventually progress into S phase and arrest in early S by a rad3- and cds1-dependent mechanism, most likely the intra-S checkpoint. Caffeine alleviates both the intra-G1- and intra-S-phase delays. We suggest that intra-G1 delay may be widely conserved and discuss significance and possible mechanisms.

Adenosine Triphosphatases↗
PubMed2002

Physical and functional interactions among basic chromosome organizational features govern early steps of meiotic chiasma formation.

Analysis of meiotic recombination by functional genomic approaches reveals prominent spatial and functional interactions among diverse organizational determinants. Recombination occurs between chromatin loop sequences; however, these sequences are spatially tethered to underlying chromosome axes via their recombinosomes. Meiotic chromosomal protein, Red1, localizes to chromosome axes; however, Red1 loading is modulated by R/G-bands isochores and thus by bulk chromatin state. Recombination is also modulated by isochore determinants: R-bands differentially favor double-strand break (DSB) formation but disfavor subsequent loading of meiotic RecA homolog, Dmc1. Red1 promotes DSB formation in both R- and G-bands and then promotes Dmc1 loading, specifically counteracting disfavoring R-band effects. These complexities are discussed in the context of chiasma formation as a series of coordinated local changes at the DNA and chromosome-axis levels.

Cell Cycle Proteins↗
PubMed2002

ATR homolog Mec1 promotes fork progression, thus averting breaks in replication slow zones.

Budding yeast Mec1, homolog of mammalian ATR, is an essential protein that mediates S-phase checkpoint responses and meiotic recombination. Elimination of Mec1 function leads to genomewide fork stalling followed by chromosome breakage. Breaks do not result from stochastic collapse of stalled forks or other incidental lesions; instead, they occur in specific regions of the genome during a G2 chromosomal transition. Break regions are found to be genetically encoded replication slow zones (RSZs), a newly discovered yeast chromosomal determinant. Thus, Mec1 has important functions in normal S phase and the genome instability of mec1 (and, analogously, ATR-/-) mutants stems from defects in these basic roles.

Chromosome Breakage↗
PubMed2002

Capturing chromosome conformation.

We describe an approach to detect the frequency of interaction between any two genomic loci. Generation of a matrix of interaction frequencies between sites on the same or different chromosomes reveals their relative spatial disposition and provides information about the physical properties of the chromatin fiber. This methodology can be applied to the spatial organization of entire genomes in organisms from bacteria to human. Using the yeast Saccharomyces cerevisiae, we could confirm known qualitative features of chromosome organization within the nucleus and dynamic changes in that organization during meiosis. We also analyzed yeast chromosome III at the G1 stage of the cell cycle. We found that chromatin is highly flexible throughout. Furthermore, functionally distinct AT- and GC-rich domains were found to exhibit different conformations, and a population-average 3D model of chromosome III could be determined. Chromosome III emerges as a contorted ring.

AT Rich Sequence↗
PubMed2002

The SUMO-1 isopeptidase Smt4 is linked to centromeric cohesion through SUMO-1 modification of DNA topoisomerase II.

In S. cerevisiae, posttranslational modification by the ubiquitin-like Smt3/SUMO-1 protein is essential for survival, but functions and cellular targets for this modification are largely unknown. We find that one function associated with the Smt3/SUMO-1 isopeptidase Smt4 is to control chromosome cohesion at centromeric regions and that a key Smt3/SUMO-1 substrate underlying this function is Top2, DNA Topoisomerase II. Top2 modification by Smt3/SUMO-1 is misregulated in smt4 strains, and top2 mutants resistant to Smt3/SUMO-1 modification suppress the smt4 cohesion defect. top2 mutants display aberrant chromatid stretching at the centromere in response to mitotic spindle tension and altered chromatid reassociation following microtubule depolymerization. These results suggest Top2 modification by Smt3/SUMO-1 regulates a component of chromatin structure or topology required for centromeric cohesion.

Cell Cycle Proteins↗