Abstract
The T4 bacteriophage Dda helicase is believed to be involved in early events in T4 DNA replication and has been shown to stimulate genetic recombination processes in vitro. Dda unwinds double-stranded DNA with 5' to 3' polarity but its ability to translocate on DNA has not been established. The DNA stimulated ATPase activity of Dda helicase has been used to probe translocation on single-strand DNA (ssDNA). Dda exhibits higher ATPase activity in the presence of poly(dT) than oligo(dT)16, indicating that Dda translocates on ssDNA. Oligonucleotides containing biotin/streptavidin blocks on the 5' or 3' end were used to probe directionality of translocation. The Kact (Km for DNA) for Dda ATPase activity was reduced in the presence of a streptavidin block on the 3' end, whereas a streptavidin block on the 5' end had only a small effect on the steady-state ATPase parameters. These results suggest that Dda translocates unidirectionally in a 5' to 3' manner and upon encountering the block remains bound to the oligonucleotide rather than sliding off the 3' end. The direction of translocation on ssDNA is consistent with the direction in which Dda unwinds duplex DNA and is not dependent on duplex structure.
Highlights
The mechanism of dsDNA unwinding by the helicase may be related to its mechanism of translocation on single-strand DNA (ssDNA)
The proposed mechanism for E. coli Rep helicase does not necessarily require unidirectional translocation on ssDNA but relies on the polarity of the ss/dsDNA junction to provide directionality in dsDNA unwinding. von Hippel and co-workers (Geiselmann et al, 1993) have proposed a model for E. coli Rho helicase in which the enzyme translocates with a 5Ј to 3Ј directional bias and “unzips” an RNA/DNA hybrid
DNA unwinding was carried out using a gel assay in which the ssDNA product was trapped after quenching the reaction by adding an excess of an unlabeled strand which prevents reannealing of product (Raney et al, 1994)
Summary
DNA, Oligonucleotides, and Reagents—Oligonucleotide substrates were synthesized by Operon Technologies (Alameda, CA) and purified by preparative gel electrophoresis. DNA was added to the helicase assay buffer and unwinding was initiated by adding Dda. The final concentration of reagents in the unwinding reactions was 25 mM Tris-OAc, pH 7.4, 10 mM Mg(OAc) mM KOAc, and 3 mM ATP in a final volume of 20 l. Reactions were stopped after 10 s by adding 20 l of quench solution containing 0.5 M EDTA and a 30-mer oligonucleotide identical in sequence to the duplex region of the labeled strand of the substrate. The latter served as a trap to prevent labeled product from reannealing with its complementary strand. The excitation wavelength for Dda was 281 nm and the emission wavelength 341 nm
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