Book of Abstracts: Albany 2003
June 17-21 2003
Ligand-induced DNA Polymerase Pausing on Double-stranded DNA
The clamp-like pyrimidine PNA oligomers, known as bisPNAs, invade the DNA double helix forming DNA-PNA2 triplexes with one DNA strands and locally displacing the other strand. When a pair of bisPNAs bind to two closely located sites on double-stranded (ds) DNA, the displaced DNA strand can hybridize with an oligonucleotide probe forming the PD-loop (1). This construct can serve as an artificial primosome: it allows the DNA polymerase with strand-displacement ability to isothermally perform the primer-extension reaction on dsDNA (2).
Using artificial primosome as a tool, we now investigate the influence of DNA-binding ligands on the DNA polymerase movement along dsDNA (see cartoon in Fig. 1 explaining our experimental design). With this approach and using an automated DNA sequencer, we analyzed several prokaryotic DNA polymerases on the ability to function on dsDNA targeted by four intercalating and groove-binding ligands: actinomycin D, chromomycin A3, distamycin A and netropsin. In our test system, two enzymes, Sequenase 2.0 and ø29 DNA polymerase, experienced multiple sequence-specific pauses during their movement along dsDNA in the presence of ligands.
Figure 1: Artificial primosome has been sequence-specifically assembled within linear, non-supercoiled dsDNA with the aid of a pair of bisPNA openers. Using DNA polymerase with the strand-displacement ability, this PNA-based construct makes it possible to study the sequence-specific pausing of DNA polymerase on duplex DNA caused by DNA-binding ligands.
For both enzymes, Sequenase 2.0 and ø29 DNA polymerase, we found that each ligand yielded its own characteristic pausing pattern for a particular DNA sequence. The majority of pausing sites could be located with a single-nucleotide resolution and corresponded to the preferred binding sites known from literature for the ligands under study. The high-quality ligand-induced pausing patterns we obtained for the first time for DNA polymerase moving along duplex DNA may become a valuable addition to the existing arsenal of methods used to monitor duplex DNA interactions with various DNA-targeting agents, including drugs. Our data also shed a new light on the DNA polymerase functioning (3).
Irina V. Smolina*
Center for Advanced Biotechnology