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- Protein Kinase A is required for chromosomal DNA replication...Protein Kinase A is required for chromosomal DNA replication
Current Biology, Volume 9, Issue 16, 26 August 1999, Pages 903-S2
Vincenzo Costanzo, Enrico V. Avvedimento, Max E. Gottesman, Jean Gautier and Domenico Grieco
SummaryPassage through mitosis resets cells for a new round of chromosomal DNA replication . In late mitosis, the pre-replication complex – which includes the origin recognition complex (ORC), Cdc6 and the minichromosome maintenance (MCM) proteins – binds chromatin as a pre-requisite for DNA replication. S-phase-promoting cyclin-dependent kinases (Cdks) and the kinase Dbf4–Cdc7 then act to initiate replication. Before the onset of replication Cdc6 dissociates from chromatin. S-phase and M-phase Cdks block the formation of a new pre-replication complex, preventing DNA over-replication during the S, G2 and M phases of the cell cycle . The nuclear membrane also contributes to limit genome replication to once per cell cycle . Thus, at the end of M phase, nuclear membrane breakdown and the collapse of Cdk activity reset cells for a new round of chromosomal replication. We showed previously that protein kinase A (PKA) activity oscillates during the cell cycle in egg extracts, peaking in late mitosis. The oscillations are induced by the M-phase-promoting Cdk . Here, we found that PKA oscillation was required for the following phase of DNA replication. PKA activity was needed from mitosis exit to the formation of the nuclear envelope. PKA was not required for the assembly of ORC2, Cdc6 and MCM3 onto chromatin. Inhibition of PKA activity, however, blocked the release of Cdc6 from chromatin and subsequent DNA replication. These data suggest that PKA activation in late M phase is required for the following S phase.
Summary | Full Text | PDF (97 kb) - Orientation of the Essential Light Chain Region of Myosin in...Orientation of the Essential Light Chain Region of Myosin in Relaxed, Active, and Rigor Muscle
Biophysical Journal, Volume 95, Issue 8, 15 October 2008, Pages 3882-3891
Andrea C. Knowles, Roisean E. Ferguson, Birgit D. Brandmeier, Yin-Biao Sun, David R. Trentham and Malcolm Irving
AbstractThe orientation of the ELC region of myosin in skeletal muscle was determined by polarized fluorescence from ELC mutants in which pairs of introduced cysteines were cross-linked by BSR. The purified ELC-BSRs were exchanged for native ELC in demembranated fibers from rabbit psoas muscle using a trifluoperazine-based protocol that preserved fiber function. In the absence of MgATP (in rigor) the ELC orientation distribution was narrow; in terms of crystallographic structures of the myosin head, the LCD long axis linking heavy-chain residues 707 and 843 makes an angle () of 120–125° with the filament axis. This is ∼30° larger than the broader distribution determined previously from RLC probes, suggesting that, relative to crystallographic structures, the LCD is bent between its ELC and RLC regions in rigor muscle. The ELC orientation distribution in relaxed muscle had two broad peaks with ∼70° and ∼110°, which may correspond to the two head regions of each myosin molecule, in contrast with the single broad distribution of the RLC region in relaxed muscle. During isometric contraction the ELC orientation distribution peaked at ∼105°, similar to that determined previously for the RLC region.
Abstract | Full Text | PDF (536 kb) - Bifunctional Rhodamine Probes of Myosin Regulatory Light Cha...Bifunctional Rhodamine Probes of Myosin Regulatory Light Chain Orientation in Relaxed Skeletal Muscle Fibers
Biophysical Journal, Volume 86, Issue 4, 1 April 2004, Pages 2329-2341
Andrew S. Brack, Birgit D. Brandmeier, Roisean E. Ferguson, Susan Criddle, Robert E. Dale and Malcolm Irving
AbstractThe orientation of the regulatory light chain (RLC) region of the myosin heads in relaxed skinned fibers from rabbit psoas muscle was investigated by polarized fluorescence from bifunctional rhodamine (BR) probes cross-linking pairs of cysteine residues introduced into the RLC. Pure 1:1 BR-RLC complexes were exchanged into single muscle fibers in EDTA rigor solution for 30min at 30°C; ∼60% of the native RLC was removed and stoichiometrically replaced by BR-RLC, and >85% of the BR-RLC was located in the sarcomeric A-bands. The second- and fourth-rank order parameters of the orientation distributions of BR dipoles linking RLC cysteine pairs 100-108, 100-113, 108-113, and 104-115 were calculated from polarized fluorescence intensities, and used to determine the smoothest RLC orientation distribution—the maximum entropy distribution—consistent with the polarized fluorescence data. Maximum entropy distributions in relaxed muscle were relatively broad. At the peak of the distribution, the “lever” axis, linking Cys and Lys of the myosin heavy chain, was at 70–80° to the fiber axis, and the “hook” helix (Pro–Lys) was almost coplanar with the fiber and lever axes. The temperature and ionic strength of the relaxing solution had small but reproducible effects on the orientation of the RLC region.
Abstract | Full Text | PDF (399 kb) - …more
Copyright © 1989 The Biophysical Society. All rights reserved.
Biophysical Journal, Volume 56, Issue 5, 927-933, 1 November 1989
doi:10.1016/S0006-3495(89)82738-9
Research Article
Disorder induced in nonoverlap myosin cross-bridges by loss of adenosine triphosphate
R. Padrón and R. Craig
Abstract
Adenosine triphosphate-dependent changes in myosin filament structure have been directly observed in whole muscle by electron microscopy of thin sections of rapidly frozen, demembranated frog sartorius specimens. In the presence of ATP the thick filaments show an ordered, helical array of cross-bridges except in the bare zone. In the absence of ATP they show two distinct appearances: in the region of overlap with actin, there is an ordered, rigorlike array of cross-bridges between the thick and thin filaments, whereas in the nonoverlap region (H-zone) the myosin heads move away from the thick filament backbone and lose their helical order. This result suggests that the presence of ATP is necessary for maintenance of the helical array of cross-bridges characteristic of the relaxed state. The primary effect of ATP removal on the myosin heads appears to be weaken their binding to the thick filament backbone; released heads that are close to an actin filament subsequently form a new actin-based, ordered array.
