DNA Replication and Cell Cycle Progression Regulatedby Long Range Interaction between Protein Complexes bound to DNA

A nonstationary interaction that controlsDNA replication and the cell cycle isderived from many-body physics in achemically open T cell. The model predictsa long range force F() =– (/2) (1 – )(2 – )between thepre-replication complexes (pre-RCs) boundby the origins in DNA, = /N being the relativedisplacement of pre-RCs, the number of pre-RCs, Nthe number of replicons to be replicated,and the compressibilitymodulus in the lattice of pre-RCs whichbehaves dynamically like an elasticallybraced string. Initiation of DNAreplication is induced at the threshold = N by a switch ofsign of F'(), fromattraction (–) and assembly in the G ₁ phase (0<<N), to repulsion (+) and partialdisassembly in the S phase (N< < 2N), withrelease of licensing factors from pre-RCs,thus explaining prevention ofre-replication. Replication is terminatedby a switch of sign of force at = 2N, from repulsion inS phase back to attraction in G ₂, when all primed replicons havebeen duplicated once. F(0) = 0corresponds to a resting cell in theabsence of driving force at = 0. The model thus ensures that the DNAcontent in G ₂ cells is exactlytwice that of G ₁ cells. The switch of interaction at the R-point, at which N pre-RCs have been assembled, starts the release of Rb protein thus also explaining the shift in the Rb phosphorylation from mitogen-dependent cyclinD to mitogen-independent cyclin E.Shape,slope and scale of the response curvesderived agree well with experimental datafrom dividing T cells and polymerising MTs,the variable length of which is due to anonlinear dependence of the growthamplitude on the initial concentrations oftubulin dimers and guanosine-tri-phosphate(GTP). The model also explains the dynamic instabilityin growing MTs.     

Long Range Force between Pre-Replication Complexes (Pre-RC) in DNA Controls Replication and Cell Cycle Progression

A nonstationary interaction, that controls DNA replication and the cell cycle, is derived from a manybody physics model in a chemically open T cell. The model predicts a long range force F()=-(/2) (1-)(2-) between the pre-replication complexes (pre-RCs) bound by DNA, =/N being the relative displacement of preRCs, the number of pre-RCs, N the threshold for initiation, and the compressibility modulus in thelattice of pre-RCs which behaves like an elastically braced string. Initiation of DNA replication is induced by a switch of sign of F(), from attraction (-)and assembly in the G ₁ phase (0 < < N), to repulsion (+) and partialdisassembly in the S phase (N < < 2N), with release of licensing factors from the pre-RCs, thus explaining prevention of re-replication. Replication is terminated by a switch of sign of F at = 2N, when all primed replicons are duplicated once, and F(0)=0 corresponds to a resting cell in absence of driving force at = 0. The switch of sign of force at = N also explains the dynamic instability in growing microtubules (MTs), as well as switch in the interleukin-2 (IL2) interaction with its receptor in late G ₁, at the restriction point. Shape, slope and scale of the response curves derived agree well with experimental data from dividing T cells and polymerizing MTs, the variable length of which is due to anonlinear dependence of the growth amplitude on the initial concentrations of tubulin dimers and guanosine-tri-phosphate (GTP).     

Myosin Motors Drive Long Range Alignment of Actin Filaments

The bulk alignment of actin filament sliding movement, powered by randomly oriented myosin molecules, has been observed and studied using an in vitro motility assay. The well established, actin filament gliding assay is a minimal experimental system for studying actomyosin motility. Here, we show that when the assay is performed at densities of actin filaments approaching those found in living cells, filament gliding takes up a preferred orientation. The oriented patterns of movement that we have observed extend over a length scale of 10–100 μm, similar to the size of a mammalian cell. We studied the process of filament alignment and found that it depends critically upon filament length and density. We developed a simple quantitative measure of filament sliding orientation and this enabled us to follow the time course of alignment and the formation and disappearance of oriented domains. Domains of oriented filaments formed spontaneously and were separated by distinct boundaries. The pattern of the domain structures changed on the time scale of several seconds and the collision of neighboring domains led to emergence of new patterns. Our results indicate that actin filament crowding may play an important role in structuring the leading edge of migrating cells. Filament alignment due to near-neighbor mechanical interactions can propagate over a length scale of several microns; much greater than the size of individual filaments and analogous to a log drive. Self-alignment of actin filaments may make an important contribution to cell polarity and provide a mechanism by which cell migration direction responds to chemical cues.     

Long Range Communication between Exosites 1 and 2 Modulates Thrombin Function

Although exosites 1 and 2 regulate thrombin activity by binding substrates and cofactors and by allosterically modulating the active site, it is unclear whether there is direct allosteric linkage between the two exosites. To begin to address this, we first titrated a thrombin variant fluorescently labeled at exosite 1 with exosite 2 ligands, HD22 (a DNA aptamer), γ′-peptide (an analog of the COOH terminus of the γ′-chain of fibrinogen) or heparin. Concentration-dependent and saturable changes in fluorescence were elicited, supporting inter-exosite linkage. To explore the functional consequences of this phenomenon, we evaluated the capacity of exosite 2 ligands to inhibit thrombin binding to γ_A/γ_A-fibrin, an interaction mediated solely by exosite 1. When γ_A/γ_A-fibrinogen was clotted with thrombin in the presence of HD22, γ′-peptide, or prothrombin fragment 2 there was a dose-dependent and saturable decrease in thrombin binding to the resultant fibrin clots. Furthermore, HD22 reduced the affinity of thrombin for γ_A/γ_A-fibrin 6-fold and accelerated the dissociation of thrombin from preformed γ_A/γ_A-fibrin clots. Similar responses were obtained when surface plasmon resonance was used to monitor the interaction of thrombin with γ_A/γ_A-fibrinogen or fibrin. There is bidirectional communication between the exosites, because exosite 1 ligands, HD1 (a DNA aptamer) or hirudin-(54–65) (an analog of the COOH terminus of hirudin), inhibited the exosite 2-mediated interaction of thrombin with immobilized γ′-peptide. These findings provide evidence for long range allosteric linkage between exosites 1 and 2 on thrombin, revealing further complexity to the mechanisms of thrombin regulation.As the central effector of hemostasis, thrombin is engaged in procoagulant, anticoagulant, and fibrinolytic processes. These seemingly contrasting roles are regulated, at least in part, by thrombin''s interactions with other factors in the blood and vasculature. The binding of ligands to thrombin is promoted by exosites 1 and 2, which are positively charged domains that flank the active site. These exosites facilitate the binding of substrates or cofactors and align them for optimal interaction with the active site (1).Exosite 1 is predominantly used to gain access to the active site by substrates such as fibrinogen (2), factors V (3) and VIII (4), and the protease-activated receptors (PARs)² on platelets (5). Effectors that modulate thrombin activity, including thrombomodulin (6), hirudin (7), and heparin cofactor II (8), also utilize exosite 1. Thrombomodulin alters the specificity of thrombin by hindering access of other substrates to exosite 1 (9) and by providing new binding sites for protein C and thrombin-activable fibrinolysis inhibitor, thereby promoting anticoagulant and antifibrinolytic pathways, respectively (10, 11). Fewer processes are mediated by exosite 2, which serves largely as a tether that anchors thrombin for participation in other reactions. Thus, heparin binds exosite 2 (12) and catalyzes thrombin inhibition by antithrombin and heparin cofactor II (13, 14). Exosite 2 also is used by glycoprotein 1bα on platelets to localize thrombin for activation of PARs (15–17).Although the prevailing role of the exosites is to bring substrates and cofactors into proximity with thrombin, there is evidence that the exosites also serve as allosteric regulators of thrombin activity. Crystallographic studies reveal that, when peptides derived from PAR1 or PAR3 are bound to exosite 1 on thrombin, an obstructing surface loop moves out of the active site pocket, thereby providing access to substrates (18). The binding of a thrombomodulin fragment to exosite 1 was shown to alter the environment of an active site fluorescent probe (19), which accelerates the rate of protein C and thrombin-activable fibrinolysis inhibitor activation in an allosteric fashion. In contrast, exosite 1-binding peptides from heparin cofactor II or fibrinogen decrease the rate of protein C activation (20). Additionally, the binding of ligands to exosite 1 alters the rates of chromogenic substrate hydrolysis (21, 22). Allosteric effects are not limited to exosite 1, because prothrombin fragment 2 (F2), a cleavage product of prothrombin, binds exosite 2 and decreases the rate at which thrombin converts fibrinogen to fibrin (23, 24) and is inhibited by antithrombin (25, 26). In support of the concept that these alterations are allosteric in origin, fluorescent probes bound to the active site of thrombin undergo a change in fluorescence intensity when exosite 2 is occupied (24, 27).Although there is good evidence for allosteric regulation of the active site by the exosites, it remains unclear whether there is direct allosteric connection between the exosites. Reciprocal effects between exosites 1 and 2 have been observed by some investigators (28–30), but not by others (25, 31). The aim of the current study was to use different techniques and additional ligands to resolve this controversy. First, we examined the effect of exosite 2-directed ligands on the fluorescence intensity of a thrombin variant that was labeled in exosite 1. Next, we examined the effect of these ligands on thrombin binding to fibrin. To exploit the observation that thrombin binds γ_A/γ_A-fibrinogen exclusively via exosite 1 (2, 32), leaving exosite 2 accessible, this subpopulation was isolated (32). We used intact fibrin clots and surface plasmon resonance (SPR) to examine the influence of exosite 2-directed ligands on thrombin binding to γ_A/γ_A-fibrin. In addition, diffusion studies were performed to examine the effect of exosite-directed ligands on the rate of thrombin dissociation from preformed fibrin clots. Finally, we explored whether exosite 1-directed ligands modulate the binding of thrombin to an exosite 2-directed ligand.     

Cis-Effects of Heterochromatin on Heterochromatic and Euchromatic Gene Activity in Drosophila Melanogaster

Chromosomal rearrangements that juxtapose heterochromatin and euchromatin can result in mosaic inactivation of heterochromatic and euchromatic genes. This phenomenon, position effect variegation (PEV), suggests that heterochromatic and euchromatic genes differ in their regulatory requirements. This report describes a novel method for mapping regions required for heterochromatic genes, and those that induce PEV of a euchromatic gene. P transposase mutagenesis was used to generate derivatives of a translocation that variegated for the light(+) (lt(+)) gene and carried the euchromatic white(+) (w(+)) gene on a transposon near the heterochromatin-euchromatin junction. Cytogenetic and genetic analyses of the derivatives showed that P mutagenesis resulted in deletions of several megabases of heterochromatin. Genetic and molecular studies showed that the derivatives shared a euchromatic breakpoint but differed in their heterochromatic breakpoint and their effects on seven heterochromatic genes and the w(+) gene. Heterochromatic genes differed in their response to deletions. The lt(+) gene was sensitive to the amount of heterochromatin at the breakpoint but the heterochromatic 40Fa gene was not. The severity of variegated w(+) phenotype did not depend on the amount of heterochromatin in cis, but varied with local heterochromatic environment. These data are relevant for considering mechanisms of PEV of both heterochromatic and euchromatic genes.     

Control of Plasmid R1 Replication: Functions Involved in Replication, Copy Number Control, Incompatibility, and Switch-off of Replication

A small derivative of plasmid R1 was used to integratively suppress a chromosomal dnaA(Ts) mutation. The strain obtained grew normally at 42°C. The integratively suppressed strain was used as recipient for various plasmid R1 derivatives. Plasmid R1 and miniplasmid derivatives of R1 could be established in the strain that carried an integrated R1 replicon, but they were rapidly lost during growth. However, plasmids also carrying ColE1 replication functions were almost completely stably inherited. The integratively suppressed strain therefore allows the establishment of bacteria diploid with respect to plasmid R1 and forms a useful and sensitive system for studies of interaction between plasmid R1 replication functions. Several of the chimeric plasmids caused inhibition of growth at high temperatures. All plasmids that inhibited growth carried one particular PstI fragment from plasmid R1 (the PstI F fragment), and in all cases the growth inhibition could be ascribed to repression of initiation of chromosome replication at 42°C, i.e., they carry a trans-acting switch-off function. Furthermore, the analogous PstI fragments from different copy mutants of plasmid R1 were analyzed similarly, and one mutant was found to lack the switch-off function. The different chimeric plasmids were also tested for their incompatibility properties. All plasmids that carried the switch-off function (and no other plasmids) also carried R1 incompatibility gene(s). Since the PstI F fragment, which is present on all these plasmids, is very small (0.35 × 10⁶), it is suggested that the switch-off regulation of replication (by an inhibitor), incompatibility, and copy number control are governed by the same gene.     

Influence of Medium and Long Range Interactions in Different Structural Classes of Globular Proteins

An analysis of the dependence known three dimensional structure ofglobular proteins on their residue contacts and their interactions providesmuch information about their folding and stability. In this work, we analysethe residue-residue contacts and the role of medium and long rangeinteractions in globular proteins belonging to different structural classes.The results show that while medium range interactions predominate in allalpha class proteins, long range interactions predominate in all beta class.The residues Pro and Gly are found to have lowest medium range contacts,probably due to their helix breaking tendency. The hydrophobic residues Ile,Val and Tyr have higher long range contacts, and hence may serve as goodnucleation centres. Further, the role of charged residues and disulfidebridges in these interactions are also discussed.