Abstract Mirkin This project focuses on the role of DNA triplexes in DNA replication. We will study homopurine-homopyrimidine sequences with varying extents of mirror symmetry that form intramolecular H-DNA triplexes in vitro. We have recently shown that these sequences cause purified DNA polymerase to terminate in double- or single-stranded DNA templates. Termination sites precisely match triplex junctions defined by chemical probing. Therefore, we suggest that the formation of triplexes prior or during DNA polymerization is responsible for premature termination of DNA synthesis. We have also found that certain orientations and positions of these sequences severely affect the maintenance of bacterial plasmids in vivo. The specific aim of this project is to prove that the formation of triplexes inhibits DNA replication both in vitro and in vivo. We will study the influence of DNA triplexes on DNA synthesis in the reconstituted replication system of bacteriophage T7 and analyze the effects of accessory replication proteins, including single-stranded DNA-binding protein and helicase-primase, on triplex-caused termination. We will characterize the formation of H-like DNA structures in E.coli cells by chemical modification of intracellular DNA followed by sequence level detection of modified bases using Maxam-Gilbert sequencing. We will measure the repression caused by cloned triplex-forming DNA sequences of the replication of the pBluescript plasmid in E.coli cells by assaying the rate of replication by the incorporation of radiolabeled precursors into DNA. We will then visualize the fine pattern of replication fork movement by two-dimensional gel electrophoresis. The long term goal of this work is to reveal the mechanisms of the inhibitory effect of triplexes on DNA synthesis in vitro and to understand the role of these structures in the regulation of DNA replication in vivo. %%% DNA usually exists as a matched pair of long molecules in a structure called double- stranded or duplex DNA. However, particular DNA segments may form an unusual structure where three, rather than two, DNA strands interact with each other. This structure is called triplex DNA. We have found that the purified cellular machinery necessary to duplicate DNA can not efficiently pass through triplex structures within DNA, leading to a block replication. Such an effect could be used to selectively inhibit the multiplication of particular DNAs such as bacteria or viruses. This aims to elucidate the inhibitory effects of triplexes on DNA replication, both in test tubes and in living cells. ***
