Department of Molecular & Cellular Biology
tel: (617) 495-4396; fax: (617) 495-0758
Mechanisms and Principles of Chromosome Activity
We are interested in the fundamental processes that govern chromosomal activities.
(1) Mechanism and regulation of transposition.Transposable elements move from place to place in genomes via a series of specific DNA breaking and joining events. We are analyzing the biochemical mechanism of transposition for a bacterial transposon, Tn10, which utilizes its own transposase protein in conjunction with accessory host factors. We wish to understand the precise nature and order of reaction steps, the types of DNA/protein complexes involved, the process by which the two ends of the transposon identify one another, the mechanistic role of host factors, the importance of DNA topology and the functional organization of transposase protein.
Chalmers, R., Guhathakurta, A., Benjamin, H. and Kleckner, N. (1998). IHF modulation of Tn10 transposition in vitro and in vivo: Sensory transduction of supercoiling status via a proposed protein/DNA molecular spring. Cell 93:897-908.
(2) Chromosome pairing and recombination during meiosis.During sexual reproduction, gametogenesis involves the halving of the cellular chromosome complement via the process of meiosis. This process requires that homologous (maternal and paternal) chromosomes recognize one another, pair along their lengths and undergo recombination. The formation of recombinants is tightly regulated. We are analyzing all of these events at the molecular level by (a) developing new assays for the processes involved, (b) identifying new genes involved in these processes, (c) carrying out biochemical analysis of relevant proteins and (d) formulating new conceptual framewords for understanding how these processes might occur.
Hunter, N. and Kleckner, N. (2001). The single-end invasion: an asymmetric intermediate at the double-strand break to double-Holliday junction transition of meiotic recombination. Cell 106: 59-70.
Zickler, D. and Kleckner, N. (1999). Meiotic chromosomes: integrating structure and function. Ann. Rev. Genet. 33: 603-754.
Blat, Y. and Kleckner, N. (1999). Cohesins bind to preferential sites along yeast chromosome III, with differential regulation along arms versus the centric region. Cell 98: 249-259.
Keeney, S., Giroux, C.N. and Kleckner, N. (1997). Meiosis-specific DNA double-strand breaks are catalyzed by Spo11, a member of a widely conserved protein family. Cell 88: 375-384.
Kleckner, N. (1996). Meiosis: how could it work? Proc. Natl. Acad. Sci. USA 93: 8167-8174.
(3) Coordination of DNA replication and cell division during the E.coli cell cycle.
In E.coli, as in most higher organisms, replication initiation
is tightly regulated. It must occur at precisely the appropriate time
in the "cell cycle" and at that time must occur once and only
once. We are investigating how this regulation is achieved. We are exploiting
our recently-developed "baby cell" machine which provides synchronous
cultures whose progression can be monitored over time. Events of the replication
and division processes are monitored by fluorescence in situ hybridization,
flow cytometry, and GFP-tagging of involved proteins. The effects of classical
and newly isolated mutations which affect these processes are being explored
in the context of new ideas for the basic logic of the bacterial program.
Lu, J., Campbell, J., Boye, E. and Kleckner, N. (1994). SeqA: a negative modulator of replication initiation in E.coli. Cell 77:413-426.
(4) Chromosome breathing.
We have recently found that chromosomes undergo cyclic expansion and contraction during both the mitotic and meiotic programs. Additional evidence suggests that expansion occurs in such a way that various constraining features are put under stress.; correspondingly, chromosome contraction tends to alleviate such stresses. We have proposed that expansional stress and stress relief govern many, perhaps all, chromosomal activities (Kleckner, Jones, Hutchinson, Dekker, Henle, Padmore and Zickler, in preparation). Future work will address key predictions of this model. An important tool in this analysis will be our newly-developed high throughput method for trapping and measuring interactions between defined DNA/chromatin fiber segments in intact nuclei (J. Dekker, unpublished). This approach provides information regarding relative spatial positioning, provides a numerical value for chromatin fiber stiffness (persistence length) and reveals the overall three-dimensional shape of whole chromosomes and, potentially, entire genomes.
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