Inhibition of Klenow fragment DNA polymerase on double-helical templates by oligonucleotide-directed triple-helix formation
We have examined the capacity of oligonucleotide-directed triple helices to block the progress of primer extension by DNA polymerase. Occupancy of the major groove of a double-helical DNA substrate obstructed Klenow fragment progress at sites that map near the proximal boundary between duplex and triplex. Among a family of related third-strand oligonucleotides that all stably occupied the target duplex in the absence of polymerase, those forming longer triplexes were more effective polymerase inhibitors than shorter complexes. Kinetic analysis revealed that the triple-helical complex provided an effective blockade for times of at least 20 min. These observations provide the basis for considering and further dissecting repair DNA polymerase function and mechanism by using various defined local three-stranded DNA structures as probes.
© 1994 American Chemical Society. Received January 7, 1994; Revised Manuscript Received March 10, 1994. Abstract published in Advance ACS Abstracts, April 15, 1994. Supported by a predoctoral fellowship from the Howard Hughes Medical Institute to J.G.H. and by research grants from the NIH (GM35924) to P.B.D. and from the NIH (AR40780) to B.J.W. We gratefully acknowledge J. Campbell for valuable discussion and critical reading of the manuscript.