Linker insertion mutations in the adenovirus preterminal protein that affect DNA replication activity in vivo and in vitro.

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RESUMO

Eighteen linker insertion mutants with mutations in the adenovirus precursor to terminal protein (pTP), which were originally constructed and tested in virions by Freimuth and Ginsberg (Proc. Natl. Acad. Sci. USA 83:7816-7820, 1986), were transferred to expression plasmids for assay of the various functions of the isolated pTP. Function was measured by the ability of individual pTP mutant proteins to participate in the initiation of replication from an adenovirus DNA end, by their activity in assays of DNA elongation, and by the intracellular distribution of pTP demonstrated by indirect immunofluorescence. Ten of the 11 mutants that were active in virion formation were also functional in DNA replication reactions in extracts, while 1 had reduced function. Four mutants with mutations that were lethal to virus production were also inactive in DNA replication reactions. These four mutations are probably located at sites required for the function of pTP in DNA synthesis. Three pTP mutants with mutations that were lethal or partially defective with respect to virion formation were active in reactions requiring pTP for initiation and elongation in extracts. All three of these mutant pTPs targeted normally to the nucleus, suggesting a defect after this step in replication. Since pTP has been reported to bind the nuclear matrix, these pTP mutants may have mutations that define sites necessary for binding to this structure. Several mutants with mutations that lie outside the putative nuclear targeting region were aberrantly localized, suggesting either that additional domains are important in nuclear localization or that there are alterations in protein structure that affect nuclear transport for some pTP mutants.

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