DNA synthesis provides the driving force to accelerate DNA unwinding by a helicase

Abstract

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)....

Communication between subunits critical to DNA binding by hexameric helicase of bacteriophage T7

“…Recent studies suggest that DNA synthesis catalyzed by T7 DNA polymerase assists the unwinding of activity of gene 4 helicase by destabilizing the junction between dsDNA and ssDNA at a replication fork (19). Consequently, a helicase defective in unwinding a duplex by itself might be assisted by concurrent DNA synthesis catalyzed by DNA polymerase.…”

Dynamic look at DNA unwinding by a replicative helicase

“…It is possible that the presence of polymerase would affect the nonmonotonic unwinding. It was shown that T7 polymerase enhances the activity of T7 helicase (46), although it is unclear at present whether this effect is through the prevention of back-slipping.…”

Cryo-EM structure of the replisome reveals multiple interactions coordinating DNA synthesis

“…The geometry of binding between gp5/trx lead and gp4 indicates that the rate of leading-strand synthesis is limited by the rate of dsDNA unwinding by the helicase (28). DNA synthesis by gp5/trx lag is not limited by its binding to gp4, but rather by the availability of ssDNA from the replication loop (Fig.…”

“…The orientation of gp5/trx lead at the replisome helps to stabilize partially unwound dsDNA at the junction between ssDNA and dsDNA, thus helping to separate dsDNA. Additionally, gp5/trx lead binding to gp4 can change the equilibrium between the forward and the backward Brownian sliding of DNA helicase along ssDNA (28,29), favoring the movement of gp4 in the 5′-3′ direction or sliding of the lagging-strand ssDNA through the central channel of gp4 in the 3′-5′ direction.…”