Helicases are molecular motors that use the energy of NTP hydrolysis to translocate along a nucleic acid strand and catalyze reactions such as DNA unwinding. The ring-shaped helicase 1 of bacteriophage T7 translocates along single stranded (ss) DNA at a speed of 130 base per second 2 . However, T7 helicase slows down nearly 10-fold when unwinding the strands of duplex DNA 3 . Here we report that T7 DNA polymerase, unable to catalyze strand displacement DNA synthesis by itself, can increase the unwinding rate to 114 base pairs per second, bringing the helicase to similar speeds as along ssDNA. The helicase-rate stimulation depends upon the DNA synthesis rate and does not rely on specific interactions between the helicase and the polymerase. Efficient duplex DNA synthesis is achieved only by the combined action of the helicase and polymerase. The DNA polymerase depends on the unwinding activity of the helicase that provides ssDNA template. The rapid trapping of the ssDNA bases by the DNA synthesis activity of the polymerase in turn drives the helicase to move forward through duplex DNA at speeds similar to those observed along ssDNA.The DNA factory of bacteriophage T7 is one of the simplest and widely used as a model system for studying replication mechnisms 4 . The T7 replication complex, containing a helicase (T7 gp4), a DNA polymerase (T7 gp5 complexed with E. coli thioredoxin), and a ssDNA binding protein (T7 gp2.5), efficiently catalyses leading and lagging strand DNA synthesis 5 . The polymerase alone can elongate a DNA primer when the downstream DNA template is singlestranded (Fig. 1a). The average rate of DNA synthesis by T7 DNA polymerase increases in a hyperbolic manner with dNTP concentration with a K 1/2 of 11 μM and V max of 230 nt s −1 (nucleotide per second) at 18 °C (Fig. 1b), which is consistent with previous pre-steady state kinetic measurements 6 . DNA synthesis is blocked when the downstream template DNA is duplex (Fig. 1c). T7 DNA polymerase incorporates only 4 to 5 nt on the duplex template before DNA synthesis stalls. These results indicate that T7 DNA polymerase cannot unwind the duplex DNA beyond 4 to 5 bp and hence cannot catalyse strand displacement DNA synthesis.T7 helicase uses the energy of dTTP hydrolysis for translocation and unwinding of duplex DNA 3,7-9 . Using an all-or-none radiometric assay carried out under single-turnover conditions 10 , we measured the unwinding activity of T7 helicase on the 30-bp replicationCorrespondence and requests for materials should be addressed to S.S.P (patelss@umdnj.edu). Competing Interests StatementThe authors declare that they have no competing financial interests. (Fig. 2a,b). T7 helicase was preincubated with the replication substrate (Fig. 2a) in the presence of dTTP without Mg 2+ (conditions that allow assembly of the protein on the DNA, but no unwinding), and reaction was started by rapid addition of Mg 2+ . T7 helicase unwinds the replication substrate at an average rate of 9 bp s −1 in the absence of T7 DNA polymerase (Fig. 2b)....

In this study, we report a neo-conceptive three-dimensionally (3D) crossing manifold micromixer (CMM) embedded in microchannel. Fabricated by sequential processes of photolithography and two photon absorption stereolithography, this leads to a microfluidic system with a built-in micromixer in a site controlled manner. The effectiveness of CMM is investigated numerically and experimentally. Through the numerical simulation, it is estimated that a high mixing ratio of 90% can be obtained even in a channel length shorter than five times the channel width. This compares well with the conventional passive type of micromixers that have a gradual increase in mixing efficiency with the length of the channel. Furthermore, the mixing performance of the realized CMM built-in microchannel is observed by confocal microscopy.

Helicases are motor proteins that use the chemical energy of NTP hydrolysis to drive mechanical processes such as translocation and nucleic acid strand separation. Bacteriophage T7 helicase functions as a hexameric ring to drive the replication complex by separating the DNA strands during genome replication. Our studies show that T7 helicase unwinds DNA with a low processivity, and the results indicate that the low processivity is due to ring opening and helicase dissociating from the DNA during unwinding. We have measured the single-turnover kinetics of DNA unwinding and globally fit the data to a modified stepping model to obtain the unwinding parameters. The comparison of the unwinding properties of T7 helicase with its translocation properties on singlestranded (ss)DNA has provided insights into the mechanism of strand separation that is likely to be general for ring helicases. T7 helicase unwinds DNA with a rate of 15 bp͞s, which is 9-fold slower than the translocation speed along ssDNA. T7 helicase is therefore primarily an ssDNA translocase that does not directly destabilize duplex DNA. We propose that T7 helicase achieves DNA unwinding by its ability to bind ssDNA because it translocates unidirectionally, excluding the complementary strand from its central channel. The results also imply that T7 helicase by itself is not an efficient helicase and most likely becomes proficient at unwinding when it is engaged in a replication complex.H elicases are ubiquitous proteins that are involved in various DNA and RNA metabolic processes that require the separation of double-stranded (ds)DNA into single strands, the removal of secondary structures in RNA, or the dissociation of proteins from nucleic acids (1-4). To perform these functions, helicases use the chemical energy from NTP hydrolysis to drive the mechanical processes of translocation and nucleic acid strand separation. In this paper, we study the mechanism of DNA unwinding by bacteriophage T7 helicase that is involved in DNA replication.During replication, the helicase has to unwind a long stretch of DNA, and that requires the helicase to couple strandseparation activity to translocation. The mechanisms of these critical processes of the helicase reaction are largely unknown. It is becoming evident that helicases can move unidirectionally along nucleic acid and displace bonded moieties along their path without specifically interacting with these moieties (5-8). Thus, unidirectional translocation is a basic activity that helicases can perform without requiring interactions with the duplex DNA. Nucleic acid strand separation is a thermodynamically unfavorable process and it is made feasible by the binding of the helicase to the newly unwound strands. Numerous mechanisms of unwinding have been proposed (2-4, 9-13), but additional experimental data are needed to distinguish between these mechanisms. For the monomeric or dimeric helicases such as the Escherichia coli PcrA and Rep helicases (14-16), it has been proposed that unwinding occurs by an active mechani...

SARS coronavirus encodes non-structural protein 13 (nsP13), a nucleic acid helicase/NTPase belonging to superfamily 1 helicase, which efficiently unwinds both partial-duplex RNA and DNA. In this study, unwinding of DNA substrates that had different duplex lengths and 5′-overhangs was examined under single-turnover reaction conditions in the presence of excess enzyme. The amount of DNA unwound decreased significantly as the length of the duplex increased, indicating a poor in vitro processivity. However, the quantity of duplex DNA unwound increased as the length of the single-stranded 5′-tail increased for the 50-bp duplex. This enhanced processivity was also observed for duplex DNA that had a longer single-stranded gap in between. These results demonstrate that nsP13 requires the presence of a long 5′-overhang to unwind longer DNA duplexes. In addition, enhanced DNA unwinding was observed for gapped DNA substrates that had a 5′-overhang, indicating that the translocated nsP13 molecules pile up and the preceding helicase facilitate DNA unwinding. Together with the propensity of oligomer formation of nsP13 molecules, we propose that the cooperative translocation by the functionally interacting oligomers of the helicase molecules loaded onto the 5′-overhang account for the observed enhanced processivity of DNA unwinding.

Methoxypoly(ethylene glycol)-block-poly(L-lysine) dendrimer was designed to form a water-soluble complex with plasmid DNA. The copolymer was synthesized by the liquid-phase peptide synthesis method. It was characterized by 1H NMR and matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrum. Agarose gel electrophoresis and DNase I protection assay proved that this linear polymer/dendrimer block copolymer assembled spontaneously with plasmid DNA, forming a water-soluble complex which increased the stability of the complexed DNA. Atomic force microscopy of the complex was evaluated at various charge ratios showing that the copolymer/DNA complex was like a globular shape.

The outbreak of severe acute respiratory syndrome (SARS) in 2002 affected thousands of people and an efficient diagnostic system is needed for accurate detection of SARS coronavirus (SARS CoV) to prevent or limit future outbreaks. Of the several SARS CoV structural proteins, the nucleocapsid protein has been shown to be a good diagnostic marker. In this study, an ssDNA aptamer that specifically binds to SARS CoV nucleocapsid protein was isolated from a DNA library containing 45-nuceotide random sequences in the middle of an 88mer single-stranded DNA. After twelve cycles of systematic evolution of ligands by exponential enrichment (SELEX) procedure, 15 ssDNA aptamers were identified. Enzyme-linked immunosorbent assay (ELISA) analysis was then used to identify the aptamer with the highest binding affinity to the SARS CoV nucleocapsid protein. Using this approach, an ssDNA aptamer that binds to the nucleocapsid protein with a K(d) of 4.93±0.30nM was identified. Western blot analysis further demonstrated that this ssDNA aptamer could be used to efficiently detect the SARS CoV nucleocapsid protein when compared with a nucleocapsid antibody. Therefore, we believe that the selected ssDNA aptamer may be a good alternative detection probe for the rapid and sensitive detection of SARS.

The present study determined whether luteolin induces HT-29 colon cancer cell death through an antioxidant effect such as the activation of antioxidant enzymes. Luteolin decreased cell viability in human colon cancer cells (HT-29), whereas it had no effect on normal colon cells (FHC). Luteolin induced apoptosis by activating the mitochondria-mediated caspase pathway in HT-29 cells. Luteolin caused loss of the mitochondrial membrane action potential, increased mitochondrial Ca2+ level, upregulated Bax, downregulated Bcl-2, induced the release of cytochrome c from mitochondria to the cytosol, and increased the levels of the active forms of caspase-9 and caspase-3. Luteolin-induced apoptosis was accompanied by the activation of intracellular and mitochondrial reactive oxygen species scavenging through the activation of antioxidant enzymes, such as superoxide dismutase and catalase in HT-29 cells. Luteolin increased the level of reduced glutathione (GSH) and the expression of GSH synthetase, which catalyzes the second step of GSH biosynthesis. The apoptotic effect of luteolin was mediated by the activation of the mitogen-activated protein kinase signaling pathway. The present results indicate that luteolin induces apoptosis by promoting antioxidant activity and activating MAPK signaling in human colon cancer cells.

Rho is a ring-shaped hexameric motor protein that translocates along nascent mRNA transcript and terminates transcription of select genes in bacteria. Using a numerical optimization algorithm that simultaneously fits all of the presteady-state ATPase kinetic data, we determine how Rho utilizes the chemical energy of ATP hydrolysis to translocate RNA. A random hydrolysis mechanism is ruled out by the observed inhibition of ATPase in a mixed hexamer containing wt and an inactive Rho mutant. We propose a mechanism in which (1) all six subunits are catalytically competent and hydrolyze ATP sequentially, (2) translocation of RNA is driven by the weak to tight binding transition of nucleotide in the catalytic site, (3) hydrolysis is coordinated between adjacent subunits by the transmission of stress via the catalytic arginine finger, (4) hydrolysis weakens the affinity of a subunit for RNA, and (5) the slow release of inorganic phosphate is controlled by changes in circumferential stress around the ring.