Showing papers by "Nancy Kleckner published in 2016"

Interrogating the Escherichia coli cell cycle by cell dimension perturbations

Abstract: Bacteria tightly regulate and coordinate the various events in their cell cycles to duplicate themselves accurately and to control their cell sizes. Growth of Escherichia coli, in particular, follows a relation known as Schaechter’s growth law. This law says that the average cell volume scales exponentially with growth rate, with a scaling exponent equal to the time from initiation of a round of DNA replication to the cell division at which the corresponding sister chromosomes segregate. Here, we sought to test the robustness of the growth law to systematic perturbations in cell dimensions achieved by varying the expression levels of mreB and ftsZ. We found that decreasing the mreB level resulted in increased cell width, with little change in cell length, whereas decreasing the ftsZ level resulted in increased cell length. Furthermore, the time from replication termination to cell division increased with the perturbed dimension in both cases. Moreover, the growth law remained valid over a range of growth conditions and dimension perturbations. The growth law can be quantitatively interpreted as a consequence of a tight coupling of cell division to replication initiation. Thus, its robustness to perturbations in cell dimensions strongly supports models in which the timing of replication initiation governs that of cell division, and cell volume is the key phenomenological variable governing the timing of replication initiation. These conclusions are discussed in the context of our recently proposed “adder-per-origin” model, in which cells add a constant volume per origin between initiations and divide a constant time after initiation.

The challenge of evolving stable polyploidy: could an increase in “crossover interference distance” play a central role?

Abstract: Whole genome duplication is a prominent feature of many highly evolved organisms, especially plants. When duplications occur within species, they yield genomes comprising multiple identical or very similar copies of each chromosome ("autopolyploids"). Such genomes face special challenges during meiosis, the specialized cellular program that underlies gamete formation for sexual reproduction. Comparisons between newly formed (neo)-autotetraploids and fully evolved autotetraploids suggest that these challenges are solved by specific restrictions on the positions of crossover recombination events and, thus, the positions of chiasmata, which govern the segregation of homologs at the first meiotic division. We propose that a critical feature in the evolution of these more effective chiasma patterns is an increase in the effective distance of meiotic crossover interference, which plays a central role in crossover positioning. We discuss the findings in several organisms, including the recent identification of relevant genes in Arabidopsis arenosa, that support this hypothesis.

Meiotic prophase roles of Rec8 in crossover recombination and chromosome structure

Abstract: Rec8 is a prominent component of the meiotic prophase chromosome axis that mediates sister chromatid cohesion, homologous recombination and chromosome synapsis. Here, we explore the prophase roles of Rec8. (i) During the meiotic divisions, Rec8 phosphorylation mediates its separase-mediated cleavage. We show here that such cleavage plays no detectable role for chromosomal events of prophase. (ii) We have analyzed in detail three rec8 phospho-mutants, with 6, 24 or 29 alanine substitutions. A distinct 'separation of function' phenotype is revealed. In the mutants, axis formation and recombination initiation are normal, as is non-crossover recombination; in contrast, crossover (CO)-related events are defective. Moreover, the severities of these defects increase coordinately with the number of substitution mutations, consistent with the possibility that global phosphorylation of Rec8 is important for these effects. (iii) We have analyzed the roles of three kinases that phosphorylate Rec8 during prophase. Timed inhibition of Dbf4-dependent Cdc7 kinase confers defects concordant with rec8 phospho-mutant phenotypes. Inhibition of Hrr25 or Cdc5/polo-like kinase does not. Our results suggest that Rec8's prophase function, independently of cohesin cleavage, contributes to CO-specific events in conjunction with the maintenance of homolog bias at the leptotene/zygotene transition of meiotic prophase.

Recombination-Independent Recognition of DNA Homology for Repeat-Induced Point Mutation (RIP) Is Modulated by the Underlying Nucleotide Sequence

Abstract: Haploid germline nuclei of many filamentous fungi have the capacity to detect homologous nucleotide sequences present on the same or different chromosomes. Once recognized, such sequences can undergo cytosine methylation or cytosine-to-thymine mutation specifically over the extent of shared homology. In Neurospora crassa this process is known as Repeat-Induced Point mutation (RIP). Previously, we showed that RIP did not require MEI-3, the only RecA homolog in Neurospora, and that it could detect homologous trinucleotides interspersed with a matching periodicity of 11 or 12 base-pairs along participating chromosomal segments. This pattern was consistent with a mechanism of homology recognition that involved direct interactions between co-aligned double-stranded (ds) DNA molecules, where sequence-specific dsDNA/dsDNA contacts could be established using no more than one triplet per turn. In the present study we have further explored the DNA sequence requirements for RIP. In our previous work, interspersed homologies were always examined in the context of a relatively long adjoining region of perfect homology. Using a new repeat system lacking this strong interaction, we now show that interspersed homologies with overall sequence identity of only 36% can be efficiently detected by RIP in the absence of any perfect homology. Furthermore, in this new system, where the total amount of homology is near the critical threshold required for RIP, the nucleotide composition of participating DNA molecules is identified as an important factor. Our results specifically pinpoint the triplet 5'-GAC-3' as a particularly efficient unit of homology recognition. Finally, we present experimental evidence that the process of homology sensing can be uncoupled from the downstream mutation. Taken together, our results advance the notion that sequence information can be compared directly between double-stranded DNA molecules during RIP and, potentially, in other processes where homologous pairing of intact DNA molecules is observed.

Quantitative Modeling and Automated Analysis of Meiotic Recombination

Abstract: Many morphological features, in both physical and biological systems, exhibit spatial patterns that are specifically characterized by a tendency to occur with even spacing (in one, two or three dimensions). The positions of crossover (CO) recombination events along meiotic chromosomes provides an interesting biological example of such an effect (1-3). In general, mechanisms that explain such patterns may (a) be mechanically-based, (b) occur by a reaction-diffusion mechanism in which macroscopic mechanical effects are irrelevant, or (c) involve a combination of both types of effects. We have proposed that meiotic CO patterns arise by a mechanical mechanism, have developed mathematical expressions for such a process based on a particular physical system with analogous properties (the so-called "beam-film model"), and have shown that the beam-film model can very accurately explain experimental CO patterns as a function of the values of specific defined parameters (4-7). Importantly, the mathematical expressions of the beam-film model can apply quite generally to any mechanism, whether it involves mechanical components or not, as long as its logic and component features correspond to those of the beam-film system (3; below). Furthermore, via its various parameters, the beam-film model discretizes the patterning process into specific components. Thus, the model can be used to explore the theoretically predicted effects of various types of changes in the patterning process. Such predictions can expand detailed understanding of the bases for various biological effects (e.g. 2, 5). We present here a new MATLAB program that implements the mathematical expressions of the beam-film model with increased robustness and accessibility as compared to programs presented previously. As in previous versions, the presented program permits both: (i) simulation of predicted CO positions along chromosomes of a test population; and (ii) easy analysis of CO positions, both for experimental data sets and for data sets resulting from simulations. The goal of the current presentation is to make these approaches more readily accessible to a wider audience of researchers. Also, the program is easily modified, and we encourage interested users to make changes to suit their specific needs. A link to the program is available on the Kleckner laboratory web site: http://projects.iq.harvard.edu/kleckner_lab.

Pattern detection at low signal-to-noise ratio

Abstract: Methods and systems for detecting and characterizing a pattern (or patterns) of interest in a low signal-to-noise ratio (SNR) data set are disclosed. One method is a two-stage Likelihood pipeline analysis that takes advantage of the benefits of a full Likelihood analysis while providing computational tractability. The two-stage pipeline may include a first stage including the application of approximate Likelihood functions in which one or more of the following assumptions or modifications may be applied: (i) the pattern of interest and background are at a specified position in a segment of the data set under examination; (ii) the SNR is low; and (iii) measurement noise can be represented in such a form that all non-position parameters of the representation are linear with respect to the derivative of the Log Likelihood versus lambda. The second stage may include a full Likelihood analysis.

Questions and Assays.

Abstract: The Thomas Hunt Morgan Medal is awarded to an individual Genetics Society of America member for lifetime achievement in the field of genetics. It recognizes the full body of work of an exceptional geneticist. The 2016 recipient is Nancy Kleckner, who has made many significant contributions to our understanding of chromosomes and the mechanisms of inheritance. Kleckner has made seminal achievements in several different research areas, including bacterial transposition, chromosome organization, and meiosis. She has repeatedly combined traditional genetic approaches with molecular biology, microscopy, physics, and modeling-unprecedented applications of these methods at the time, but which have now become commonplace. Indeed, she is widely recognized as one of the leaders in bringing meiosis research into the modern era. Notably, her laboratory played a key role in elucidating the mechanism that initiates meiotic recombination, has helped to decipher the "strand gymnastics" of recombination, and is beginning to provide insight into the enigmatic phenomenon of crossover interference.