The molecular mechanism by which bacteriophage T7 replicates its DNA has been well characterized. All of the proteins known to have a role in the replication process have been isolated in homogeneous form and their contribution in the replication of the T7 DNA molecule have, in general, been characterized. The DNA polymerase and gene 4 protein (T7 primase) of bacteriophage T7 are able to initiate DNA synthesis at a phosphodiester bond interruption (nick) in a duplex T7 DNA molecule leading to extensive DNA synthesis. In the presence of the T7 RNA polymerase, DNA synthesis instead can initiate at the origin of T7 DNA replication which lies between nucleotide 5921 and 6049. This site-specific synthesis gives rise to replication bubbles at the primary origin as seen in the electron microscope. We propose to use this well characterized system as a model to study the effects of covalent modification of a DNA template by a carcinogen. We will (i) determine and compare the consequences of benzo[a]pyrene, aminofluorene, and acetylaminofluorene adducts on RNA synthesis by the T7 RNA polymerase, DNA synthesis by the T7 DNA polymerase and gene 4 protein, and DNA synthesis by the T7 in vitro replication system; (ii) determine, using DNA and RNA sequencing techniques, whether synthesis is blocked by these adducts and identify the specific sites of blockage; (iii) study the effects of DNA modification on the binding interactions between the T7 replication proteins and DNA templates; and (iv) determine the effect on the initiation of T7 DNA replication in vitro. These studies will use both single-stranded, duplex linear, and supercoiled DNA templates and DNA molecules which contain adducts located at specific sites and specifically incorporated into only the strand serving as the template or into only the strand which is being displaced.
