Entering and exiting DNAs

Sivolob, A., De Lucia, F., Revet, B., and Prunell, A. (1999) Nucleosome dynamics II. High flexibility of nucleosome entering and exiting DNAs to positive crossing. An ethidium bromide fluorescence study of mononucleosomes on DNA minicircles. J. Mol. Biol. 285, 1081-1099. 

Fig. 3. (A) Stereo pair of starfish sperm chromatin fibers stained with osmium ammine-B. Bracket indicates the fine deposition of stain on nucleosomes and linker DNA. Arrowheads indicate where fibers enter and exit the plane of the section, and asterisks indicate sharp bends in the fibre axis. (B) Stereo pair of a reconstructed volume of a starfish sperm head by EM tomography. Axes of some fibers have been marked. Arrowhead indicates where individual fibers cannot be distinguished (from Ref. [27]). Scale bar 100 nm.

Fig. 4. Images of unfixed and unstained chromatin in a frozen and hydrated state. All samples shown contain linker histone H5. (A) Soluble chromatin prepared from chicken erythrocyte nuclei. Arrow indicates a nucleosome with a linker histone stem conformation. (B-E) Chromatin reconstituted onto an array of the 5S rDNA nucleosome positioning sequence. En face views (B-D) of nucleosomes show the linker DNA entering and exiting the nucleosome tangentially, before interacting and remaining associated for 3-5 nm before separating (arrows). An edge-on view (E) shows the two gyres of DNA (arrow heads) and the apposed linker DNA (arrow) (from Ref. [30]). Scale bar 20 nm (A) and 10 nm (B-E).

Initial studies used cytochalasin D to disrupt the actin cytoskeleton and probe the role of p53 in subsequent entry into the cell cycle. Cells treated with cytochalasin D enter and exit mitosis, segregate their chromosomes but are unable to divide. These cells eventually exit mitosis as a binucleated G1 cell. Initial studies indicated that this tetraploid state was sufficient to induce p53 and block entry into a new S phase. Cells lacking p53 did enter S phase, duplicated their genome, and became octoploid (i.e., via endoreduphcation). More recent evidence suggests that the drug treatments themselves induce p53, and simply having a tetraploid DNA content is not sufficient for p53 induction. 

Shortly after acute renal failure many normally quiescent kidney cells enter the cell cyde. Orderly progression through the cell cycle is regulated by sequential synthesis, activation, compartmentalization and degradahon of proteins controUing both entry and exit from each of the four phases of the cyde G1 (gap-1), S (DNA synthesis), G2 (gap-2) and M (mitosis) (Figure... 

The Xenopus system has proven instrumental in determining the mechanism controlling exit from mitosis at the metaphase/anaphase transition. Studies in this area have relied heavily on extracts prepared from fully mature oocytes/ unfertilized eggs that are arrested at metaphase of the second meiotic division. Upon Ca2+ addition, anaphase is initiated and the extract enters the first embryonic cell cycle to replicate DNA. The activity responsible for metaphase arrest was discovered by Masui at the same time as MPF (Masui Markert 1971), and given the name cytostatic factor (CSF). CSF has never been purified... 

Fig. 20. Packing interactions between NCP molecules, which are a consequence of crystallization, nevertheless provide hints for higher order chromatin structure assembly, (a) Histone-histone interactions shown at the site of the cacodylate ion. In addition to binding interactions with the cacodylate ion, the N-terminal tail is involved in significant interactions with the patch of acidic residues on the dimer face of the neighbor NCP. The orientation of the dyad alternates between the two NCP molecules, (b) DNA base stacking is continuous between neighboring NCP molecules in the crystal lattice as the DNA exits one NCP and enters the next. The stacking interaction is strong enough to force a shift in the terminal phosphates for adjoining 5 termini.

Addition of a poly-A tail Most eukaryotic mRNAs (with several notable exceptions, including those coding for the histones and some interferons) have a chain of 40 to 200 adenine nucleotides attached to the 3 -end (see Rgure 30.17). This poly-A tail is not transcribed from the DNA, but rather is added after transcription by the nuclear enzyme, polyadenylate polymerase. A consensus sequence, called the polyadenylation signal sequence (AAUAAA), found near the 3 -end of the RNA molecule, signals that a poly-A tail is to be added to the mRNA. These tails help stabilize the mRNAs and facilitate their exit from the nucleus. After the mRNA enters the cytosol, the poly-A tail is gradually shortened. 

The binding of cisplatin to DNA is not in itself sufficient to cause cell death. Cells usually take several days to die after incubation with cisplatin, and it is during this time that decisions are made that impact on the survival or death of the cell. Cisplatin is generally considered a cell-cycle-phase non-specific drug, but it is still more toxic to dividing cells than to resting cells. Furthermore, cisplatin can be up to 10 times more toxic to cells that are about to enter S-phase than cells that have just exited S-phase. In one series of experiments, normal human fibroblasts were incubated with cisplatin while arrested at confluence at various times thereafter, they were analyzed for the amount of DNA-bound platinum, and plated to score cell... 

Cell cycle Is the repeated series of events In cells that lead to division of a parental cell Into tw/o daughter cells. The cell cycle Is divided Into Interphase and M phase. The Interphase encompasses the Gl phase (gap 1, cell growth), S phase (synthesis, DNA replication), G2 phase (gap 2, cell growth and preparation for mitosis). During the M phase, mitosis and cytokinesis take place. After completion of cell division, cells may enter the Gl phase for a next round ofcell division. Alternatively, cells may exit the cell cycle and remain in a resting state (also called GO phase). In this quiescent state, cells can remain for a long period of time or even indefinitely (Figure 1). 

To this end, the transloeation time depends on external factors, such as Vbias, the solution viseosity rj, temperature, analyte/surface interactions as well as the properties of the analytes itself. For a DNA molecule to trans-loeate, the strand must first be eaptured by the eleetrie field and then un-coil to enter the pore (if the pore is suffieiently small sueh that the DNA cannot pass through in a folded eonformation). After translocation, the DNA will inevitably re-coil when exiting the pore. This relaxation time, which has been found to be around 100 ms for a 10 kb dsDNA, will contribute significantly to the translocation dwell time of a DNA molecule [12]. Such effects also to some extent explain why controlling the translocation process with high spatial resolution is such a challenge [13]. 

---