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Nucleic Acids Research, 1985, Vol. 13, No. 13 4935-4952
© 1985

Articles

Adenovinis DNA replication in vitro: site-directed mutagenesis of the nuclear factor I binding site of the Ad2 origin

Erik de Vries, Wim van Driel, Marijke Tromp*, Jacques van Boom* and Peter C. van der Vliet

Laboratory for Physiological Chemistry, State University of Utrecht Vondellaan 24a, 3521 GG Utrecht *Organic Chemistry Laboratory, Gorlaeus Laboratories, State University of Leiden P.O. Box 9502, 2300 RA Leiden, The Netherlands

Received April 29, 1985. Revised June 10, 1985. Accepted June 10, 1985.

The template requirements for efficient adenovirus DNA replication were studied in vitro in a reconstituted system with cloned DNA fragments, containing the Ad2 origin region, as templates. Replication is enhanced by nuclear factor I, a cellular protein that binds specifically to the Ad2 origin. This stimulation is shown to be strongly dependent on the concentration of the adenovirus DNA binding protein. Using synthetic oligonucleotides we have constructed plasmids with base substitutions in the nuclear factor I binding region. Footprint analysis and competition filter binding studies show that two of the three small blocks of conserved nucleotides in this region are involved in the binding of nuclear factor I. The binding affinity can be influenced by the base composition of the degenerate region just outside these two blocks. In vitro initiation and DNA chain elongation experiments with the mutants demonstrate that binding of nuclear factor I to the Ad2 origin is necessary for stimulation. However, binding alone is not always sufficient since a mutation which only slightly disturbs binding is strongly impaired in stimulation of DNA replication by nuclear factor I.


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