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)....

Abstact. Meningiomas are the most commonly occurring tumors of the central nervous system including the brain and spinal cord. Malignant meningiomas are highly aggressive and frequently recur after surgical resection of the tumor. Our previous studies have reported that urokinase plasminogen activator receptor (uPAR) and matrix metalloproteinase-9 (MMP-9) play important roles in tumor progression. In the present study, we have attempted to evaluate the roles of these molecules in the malignant meningioma tumor microenvironment and to determine the effectiveness of using single or bicistronic small interfering RNA constructs for uPAR and MMP-9 on tumor cell proliferation, migration, invasion, angiogenesis and regression of pre-established orthotopic tumors. Transfection of single or bicistronic constructs downregulated uPAR and MMP-9 in meningioma cells compared to controls. A significant reduction in tumor invasion was determined with Matrigel gel and spheroid invasion assays in meningioma cells after transfection of these plasmids. Furthermore, downregulation of uPAR and MMP-9 reduced migration of tumor spheroids on vitronectin-coated plates. uPAR and MMP-9 downregulation suppressed capillary network formation, in both in vitro and in vivo models. Also, it is well known that tumor cells manipulate intracellular signaling pathways to aid in various processes involved in tumor progression. Our study revealed that downregulation of uPAR and MMP-9 leads to a decrease in the activation of some of the important enzymes participating in the MAPK and PI3 kinase pathways, which in turn, might decrease cell survival and proliferation. In addition, we analyzed the efficiency of RNAi-mediated targeting of uPAR and MMP-9 in pre-established tumor growth in vivo. We observed a significant regression of pre-established orthotopic tumors upon RNAimediated targeting of uPAR and MMP-9. In addition, the present study indicated that targeting both the proteins simultaneously augmented the therapeutic treatment of human meningiomas.

Abstract. Malignant meningiomas are highly aggressive and frequently recur after surgical resection of the tumor. Earlier studies have reported that the cysteine protease cathepsin B and the matrix metalloproteinase MMP-9 play important roles in tumor progression. In the present study, we made an attempt to evaluate the roles of these proteases in the malignant meningioma tumor microenvironment and determined the effectiveness of using single or bicistronic siRNA constructs for cathepsin B and MMP-9, in both in vitro and in vivo models. Transfection of a plasmid vector expressing doublestranded RNA for cathepsin B and MMP-9 significantly inhibited mRNA and protein levels of cathepsin B and MMP-9. The migration and invasion of meningioma cells were decreased after treatment with single or bicistronic siRNA constructs for cathepsin B and MMP-9 compared to controls and vector controls. Inhibition of angiogenesis was observed when the cells were transfected with single or bicistronic constructs for cathepsin B and MMP-9, when compared to controls or empty vector controls. Our study revealed that abrogation of cathepsin B and MMP-9 expression decreased the activation of major proteins involved in MAP kinase and PI3 kinase pathways indicating that targeting these proteases may hinder intracellular signaling and thus decrease cell survival and proliferation in malignant meningiomas. In addition to the in vitro evidence, we observed a significant regression of pre-established orthotopic tumors after treatment with RNAi plasmid vectors targeting cathepsin B and MMP-9. Furthermore, these observations demonstrate that the simultaneous RNAi-mediated targeting of cathepsin B and MMP-9 has potential application for the treatment of human meningiomas.