Chi Psi Research Articles

Microwave Performance of Flip Chip Interconnects With Anisotropic and Non-conductive Films

Anisotropic conductive films (ACFs) and non-conductive films (NCFs) are film-type, adhesive polymer resins that have been used in flat panel display modules to provide high-resolution, light weight, thin profile and environmental friendliness. We investigated the radiofrequency (RF) and high frequency performance of the flip chip interconnects with ACF and NCF by measuring the scattering parameters (S-parameters) of the flip chip modules. The effects of three chip materials, Si, quartz and gallium arsenide (GaAs), and the metal pattern gap between the signal line and ground plane in the coplanar waveguide (CPW) on the RF performance of the flip chip were also investigated. The transmission properties of the GaAs and quartz chips were markedly improved over those of the Si chip, which was not suitable for the measurement of the S-parameters of the flip chip interconnect. The RF performance of the flip chip module with NCF was slightly better than that of the module with ACF, mainly due to the capacitance of the epoxy resin between the Au bump and pad and the self-inductance of the conductive particle surface in the ACF interconnect.

Mechanism of the δ Wrench in Opening the β Sliding Clamp

The beta sliding clamp encircles DNA and tethers DNA polymerase III holoenzyme to the template for high processivity. The clamp loader, gamma complex (gamma 3 delta delta'chi psi), assembles beta around DNA in an ATP-fueled reaction. The delta subunit of the clamp loader opens the beta ring and is referred to as the wrench; ATP modulates contact between beta and delta among other functions. Crystal structures of delta.beta and the gamma 3 delta delta' minimal clamp loader make predictions of the clamp loader mechanism, which are tested in this report by mutagenesis. The delta wrench contacts beta at two sites. One site is at the beta dimer interface, where delta appears to distort the interface by via a steric clash between a helix on delta and a loop near the beta interface. The energy for this steric clash is thought to derive from the other site of interaction, in which delta binds to a hydrophobic pocket in beta. The current study demonstrates that rather than a simple steric clash with beta, delta specifically contacts beta at this site, but not through amino acid side chains, and thus is presumably mediated by peptide backbone atoms. The results also imply that the interaction of delta at the hydrophobic site on beta contributes to destabilization of the beta dimer interface rather than acting solely as a grip of delta on beta. Within the gamma complex, delta' is proposed to prevent delta from binding to beta in the absence of ATP. This report demonstrates that one or more gamma subunits also contribute to this role. The results also indicate that delta' acts as a backboard upon which the gamma subunits push to attain the ATP induced change needed for the delta wrench to bind and open the beta ring.

Carboxyl-terminal Domain III of the δ′ Subunit of DNA Polymerase III Holoenzyme Binds DnaX and Supports Cooperative DnaX Complex Assembly

The delta' subunit of the DNA polymerase-III holoenzyme is a key component of the DnaX complex; it is required for loading the beta(2) processivity factor onto a primed template. The x-ray crystal structure of delta' indicates a three domain C-shaped structure (Guenther, B., Onrust, R., Sali, A., O'Donnell, M., and Kuriyan, J. (1997) Cell 91, 335-345). In this study, we localized the DnaX-binding domain of delta' to its carboxyl-terminal domain III by quantifying protein-protein interactions using a series of delta' fusion proteins lacking specific domains. The fusion protein corresponding to domain III of delta' bound to DnaX with an affinity approaching that of full-length delta'. In contrast, a construct bearing delta' domains I-II did not bind DnaX at detectable levels. The presence of delta and chi psi strengthened the interaction of DnaX with full-length delta' and delta' domain III. Thus, domain III of delta' not only contains the DnaX-binding site, but also contains the elements required for positive cooperative assembly of the DnaX complex. A domain III-specific anti-delta' monoclonal antibody interfered with DnaX complex formation and abolished the replication activity of DNA polymerase III holoenzyme.

X-Ray Stress Tensor Analysis in a Notch with 2 Circles Omega Goniometer

X-ray tensor analysis requires diffraction acquisition in several directions described by two angles and ψ. These are usually achieved by chi (psi) and omega settings on a 3 or 4 circles goniometer In the case of a deep notch, problems of shadowing may occur, particularly with omega setting. To overcome this, we kept the diffraction plane parallel to the axis of the notch. The measurement direction was defined by two angles: the ω rotation of the goniometer and a. With the assumption of homogeneous stress state along the circumference, a was obtained by a translation of the part parallel to the ω axis. The classical relations between the measured strain and the stress tensor components was reformulated using directly the two angles a and ω. The influence of goniometer alignment and part positioning was analysed through an experiment design. Systematic errors due to the interactions of the incident beam with the surface of the notch were modelised. This method was applied to a toric groove in a quenched steel forming tool. Compressive residual stresses of-520 MPa and -980 MPa were found in the axial and hoop directions respectively.

ATP Binding to the Escherichia coli Clamp Loader Powers Opening of the Ring-shaped Clamp of DNA Polymerase III Holoenzyme

The Escherichia coli gamma complex serves as a clamp loader, catalyzing ATP-dependent assembly of beta protein clamps onto primed DNA templates during DNA replication. These ring-shaped clamps tether DNA polymerase III holoenzyme to the template, facilitating rapid and processive DNA synthesis. This report focuses on the role of ATP binding and hydrolysis catalyzed by the gamma complex during clamp loading. We show that the energy from ATP binding to gamma complex powers several initial events in the clamp loading pathway. The gamma complex (gamma2 delta delta'chi psi) binds two ATP molecules (one per gamma subunit in the complex) with high affinity (Kd = 1-2. 5 x 10(-6) M) or two adenosine 5'-O-(3-thiotriphosphate)(ATPgammaS) molecules with slightly lower affinity (Kd = 5-6.5 x 10(-6) M). Experiments performed prior to the first ATP turnover (kcat = 4 x 10(-3) s-1 at 4 degreesC), or in the presence of ATPgammaS (kcat = 1 x 10(-4) s-1 at 37 degreesC), demonstrate that upon interaction with ATP the gamma complex undergoes a change in conformation. This ATP-bound gamma complex binds beta and opens the ring at the dimer interface. Still prior to ATP hydrolysis, the composite of gamma complex and the open beta ring binds with high affinity to primer-template DNA. Thus ATP binding powers all the steps in the clamp loading pathway leading up to the assembly of a gamma complex. open beta ring.DNA intermediate, setting the stage for ring closing and turnover of the clamp loader, steps that may be linked to subsequent hydrolysis of ATP.

DnaX Complex of Escherichia coli DNA Polymerase III Holoenzyme THE χ·ψ: COMPLEX FUNCTIONS BY INCREASING THE AFFINITY OF τ AND γ FOR δ·δ′ TO A PHYSIOLOGICALLY RELEVANT RANGE

An artificial operon that contains tandem holC-holD genes was used to overproduce a complex of the chi and psi subunits of the DNA polymerase III holoenzyme. Normally insoluble by itself, psi forms a tight soluble complex with chi. A purification procedure that yields pure, active chi psi complex in 100-mg quantities suitable for biophysical studies is reported. Sedimentation equilibrium studies demonstrate that chi psi is a 1:1 heterodimer. The presence of chi psi dramatically lowers the level of delta.delta' required to reconstitute holoenzyme to levels expected in vivo. That chi psi accomplishes this by binding to gamma or tau and increasing their affinity for delta.delta' was demonstrated by surface plasmon resonance using a Pharmacia BIA-core instrument. In the absence of delta.delta', chi psi binds to either the gamma or tau DnaX protein with Kd = 2 nM.

Assembly of a Chromosomal Replication Machine: Two DNA Polymerases, a Clamp Loader, and Sliding Clamps in One Holoenzyme Particle.: IV. ATP-BINDING SITE MUTANTS IDENTIFY THE CLAMP LOADER

The gamma complex (gamma delta delta' chi psi) and tau complex (tau delta delta' chi psi) clamp loaders require ATP hydrolysis to load beta sliding clamps onto DNA. The beta sliding clamp tethers the polymerase (Pol) III* replicase to DNA for processive synthesis. Pol III* contains both gamma and tau, but only one each of the delta, delta', chi, and psi subunits. Hence, there is ambiguity with respect to which clamp loader, the gamma or tau complex, exists in the Pol III* replicase structure. In this study, ATP-binding site mutants of gamma and tau have been prepared, and these mutants, when assembled into either the gamma or tau complex, are inactive in clamp loading. These mutants have been used as a tool to determine the identity of the clamp loader in Pol III*. The nine-subunit Pol III* has been assembled using either mutant gamma or tau in place of wild-type gamma or tau. The results show that mutation of gamma inactivates Pol III* activity, but mutation of tau does not, indicating that the gamma complex (and not the tau complex) is the clamp loader of Pol III*. The tau subunit carries the task of dimerizing the core polymerase, and it is this association of tau with core that appears to direct the single copy subunits away from tau and onto gamma.

DNA polymerase III accessory proteins. II. Characterization of delta and delta'

The gamma complex subassembly (gamma delta delta' chi psi) of DNA polymerase III holoenzyme couples ATP to assemble the ring-shaped beta subunit around DNA forming a DNA sliding clamp. This beta clamp is needed for highly processive synthesis by the holoenzyme. Here, the delta and delta' subunits of the gamma complex are studied for their structural and functional interaction with each other and with the gamma subunit. Both delta and delta are monomeric in their native state, and they bind each other tightly to form a 1:1 complex. Neither delta nor delta' alone binds tightly to the gamma subunit. However, as a complex, delta delta' binds gamma tightly to form a gamma delta delta' complex. The fact that all three subunits, gamma, delta, and delta', are needed to form a tight complex correlates well with activity assays which show that gamma and delta are capable but inefficient in assembly of the beta ring onto DNA and delta' is needed for an efficient reaction.

DNA polymerase III accessory proteins. IV. Characterization of chi and psi

The gamma complex (gamma delta delta' chi psi) subassembly of the Escherichia coli replicase initiates processive replication upon assembling the ring-shaped beta subunit around DNA. The beta ring acts as a clamp to hold the replicase down to DNA for highly processive synthesis. In this report we characterize the chi and psi subunits of the gamma complex. Both chi and psi are monomeric, and they associate to form a 1:1 complex. The chi subunit does not form a complex with gamma, but psi binds gamma tightly thereby acting as a bridge to assimilate chi into the gamma complex structure. The psi subunit stimulated the ATPase and replication activities of gamma. The chi subunit only stimulated the activities of gamma when the psi subunit was also present thus reflecting the structure where psi bridges the interaction of chi with gamma.

Analysis of the ATPase subassembly which initiates processive DNA synthesis by DNA polymerase III holoenzyme.

The gamma complex (gamma delta delta' chi psi) subassembly of DNA polymerase III holoenzyme transfers the beta subunit onto primed DNA in a reaction which requires ATP hydrolysis. Once on DNA, beta is a "sliding clamp" which tethers the polymerase to DNA for highly processive synthesis. We have examined beta and the gamma complex to identify which subunit(s) hydrolyzes ATP. We find the gamma complex is a DNA dependent ATPase. The beta subunit, which lacks ATPase activity, enhances the gamma complex ATPase when primed DNA is used as an effector. Hence, the gamma complex recognizes DNA and couples ATP hydrolysis to clamp beta onto primed DNA. Study of gamma complex subunits showed no single subunit contained significant ATPase activity. However, the heterodimers, gamma delta and gamma delta', were both DNA-dependent ATPases. Only the gamma delta ATPase was stimulated by beta and was functional in transferring the beta from solution to primed DNA. Similarity in ATPase activity of DNA polymerase III holoenzyme accessory proteins to accessory proteins of phage T4 DNA polymerase and mammalian DNA polymerase delta suggests the basic strategy of chromosome duplication has been conserved throughout evolution.

Processive replication is contingent on the exonuclease subunit of DNA polymerase III holoenzyme.

In this report we have taken the reconstitution approach to study which subunits of the heterotrimer core polymerase (alpha, epsilon, theta) participate in the highly processive replication of long DNA templates by DNA polymerase III holoenzyme (holoenzyme). Comparison of the core and the alpha epsilon complex (the DNA polymerase and 3'-5' exonuclease subunits, respectively) shows they are both rapid and highly processive polymerases when they are reconstituted into a holoenzyme with the gamma complex (gamma delta delta' chi psi) and beta accessory proteins of holoenzyme. Specifically, holoenzyme reconstituted using either core or alpha epsilon completely replicates a uniquely primed 5.4-kilobase (kb) single-stranded DNA within 12 s in one binding event. Hence the theta subunit of core is not required for the processivity or speed of the holoenzyme. In contrast, when only the alpha subunit is reconstituted into the holoenzyme it is unable to replicate the entire 5.4-kb circle in one binding event but still retains a fairly high processivity of 1-3 kb and when given sufficient time for multiple binding events it finally finishes the entire circle. Therefore, highly processive DNA synthesis by holoenzyme is contingent on the epsilon exonuclease subunit. In light of these results the significance of the polymerase and exonuclease activities residing in two separate polypeptides is discussed.

Total reconstitution of DNA polymerase III holoenzyme reveals dual accessory protein clamps.

DNA polymerase III holoenzyme (holoenzyme) is the 10-subunit replicase of the Escherichia coli chromosome. In this report, pure preparations of delta, delta', and a gamma chi psi complex are resolved from the five protein gamma complex subassembly. Using these subunits and other holoenzyme subunits isolated from overproducing plasmid strains of E. coli, the rapid and highly processive holoenzyme has been reconstituted from only five pure single subunits: alpha, epsilon, gamma, delta, and beta. The preceding report showed that of the three subunits in the core polymerase, only a complex of alpha (DNA polymerase) and epsilon (3'-5' exonuclease) are required to assemble a processive holoenzyme on a template containing a preinitiation complex (Studwell, P.S., and O'Donnell, M. (1990) J. Biol. Chem. 265, 1171-1178). This report shows that of the five proteins in the gamma complex only a heterodimer of gamma and delta is required with the beta subunit to form the ATP-activated preinitiation complex with a primed template. Surprisingly, the delta' subunit does not form an active complex with gamma but forms a fully active heterodimer complex with the tau subunit (as does delta). Hence, the tau delta' and gamma delta heterodimers are fully active in the preinitiation complex reaction with beta and primed DNA. Holoenzymes reconstituted using the alpha epsilon complex, beta subunit, and either gamma delta or tau delta' are fully processive in DNA synthesis, and upon completing the template they rapidly cycle to a new primed template endowed with a preinitiation complex clamp. Since the holoenzyme molecule contains all of these accessory subunits (gamma, delta, tau, delta', and beta) in all likelihood it has the capacity to form two preinitiation complex clamps simultaneously at two primer termini. Two primer binding components within one holoenzyme may mediate its rapid cycling to multiple primers on the lagging strand and also provides functional evidence for the hypothesis of holoenzyme as a dimeric polymerase capable of simultaneous replication of both leading and lagging strands of a replication fork.

DNA Polymerase III holoenzyme of Escherichia coli. IV. The holoenzyme is an asymmetric dimer with twin active sites.

Pol III, a subassembly of Escherichia coli DNA polymerase III holoenzyme lacking only the auxiliary beta subunit, was purified to homogeneity by an improved procedure. This assembly consists of nine different polypeptides, likely in a 1:1 stoichiometry: a catalytic core (pol III) of alpha (132 kDa), epsilon (27 kDa), and theta (10 kDa), and six auxiliary subunits: tau (71 kDa), gamma (52 kDa), delta (35 kDa), delta' (33 kDa), chi (15 kDa), and psi (12 kDa). The assembly behaves on gel filtration as a particle of about 800 kDa, indicating a content of two each of the subunits. A new procedure for purifying the core yielded a novel dimeric form which may provide the foundation for the dimeric nature of the more complex pol III and holoenzyme forms. Pol III readily dissociates into several subassemblies including pol III', likely a dimeric core with two tau subunits. The holoenzyme, purified by a similar procedure with ATP and Mg2+ present throughout, retained the beta subunit (37 kDa) as well as all the subunits present in pol III; the mass of the holoenzyme was estimated to be 900 kDa. The isolated initiation complex of holoenzyme with a primed template DNA and the elongation complex (formed in the presence of three deoxynucleoside triphosphates) had the same composition and stoichiometry as observed for pol III with two beta dimers in addition. An initiation complex assembled from a mixture of monomeric pol III core, gamma 2 delta delta' chi psi complex (gamma complex), beta, and tau retained the core, one beta dimer, and two tau subunits but was deficient in the gamma complex. When tau was omitted from the assembly mixture, the initiation complex contained one or two gamma complexes instead of the tau subunit. Based on these data, pol III holoenzyme is judged to be an asymmetric dimeric particle with twin pol III core active sites and two different sets of auxiliary units designed to achieve essentially concurrent replication of both leading and lagging strand templates.