The objective is to continue to investigate the detailed effects of the bulky carcinogenic adducts, 2-aminofluorene (AF), N-acetyl-2-aminofluorene (AAF), and benzo(a)pyrene (BP), on DNA replication. Our approach has been to study the effect of these adducts on not just a DNA polymerase alone, but, instead, to determine how a purified in vitro replication system responds to this type of damage. We propose to determine how the ancillary proteins, required for DNA replication in vivo, affect synthesis using single- or double-stranded DNA templates containing carcinogenic DNA adducts and to characterize the effect of such adducts on the fidelity of DNA synthesis, DNA unwinding, RNA transcription, DNA initiation, and protein-nucleic acid interactions. The mechanism by which E. coli single stranded DNA binding protein (SSB) stimulates DNA synthesis by the T7 DNA polymerase on native and carcinogen-modified DNA will be determined and this effect will be compared with the results obtained with other DNA polymerases. These experiments will utilize not only randomly modified single- and double-stranded DNA templates, but also DNA containing a site-specific AF and AAF adducts, which we have recently prepared. Using these site-specifically modified templates, the frequency of misincorporation during trans-lesion synthesis by DNA polymerases in the presence and absence of SSB or other single-stranded DNA binding proteins will be determined. DNA synthesis by the T7 DNA polymerase and gene 4 protein on double-stranded DNA containing AF, AAF, and BP adducts will be characterized and these studies will be subsequently extended to site-specifically modified templates. DNA molecules containing a T7 class III RNA polymerase promoter and containing an AF or AAF adduct located a unique site will be prepared and used to further characterize the effect of DNA adducts on RNA synthesis and on the initiation of DNA replication by the T7 RNA polymerase, T7 DNA polymerase, and gene 4 protein. Finally, we will determine the effect of DNA adducts on the interactions between DNA and single-stranded DNA binding proteins, T7 RNA polymerase, or the Uvr ABC endonuclease.
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