Mutational analysis of single-stranded DNA templates active in the in vitro initiation assay for adenovirus DNA replication

Three distinct domains, A, the minimal origin, as well as B and C, the binding sites for the host nuclear factors, are required for efficient initiation of adenovirus (Ad) DNA replication at the termini. The initiation reaction was examined using partially purified nuclear extracts and various single-stranded oligomers as DNA templates. We observed that single-stranded oligomers containing Ad2 minimal origin (Ori) sequences (bp 1-18) from the I-strand of the Ad2 genome supported preterminal protein-dCMP complex formation in vitro. Using oligomers containing point mutations in the Ad2 minimal Ori sequence, six positions were identified as important to the function of the Ad2 minimal Ori sequence. Point mutations at position 7, 8, or 11 virtually abolished the ability of the oligomer to support the initiation reaction. Point mutations at position 4, 9, or 17 were found to decrease the ability of the oligomers to support the initiation reaction to 33, 67, and 58% of control, respectively. An oligomer complementary to the I-strand of the Ad2 minimal Ori was found to block initiation on minimal Ori template. A number of randomly selected nonspecific oligomers did not, in general, serve as templates for initiation with the exception of two oligomers, one of which was found to be about threefold more active than the control minimal Ori template. The biological significance of the in vitro initiation of Ad2 DNA replication on single-stranded DNA templates is discussed.