Adenovirus (Ad) DNA synthesis will be studied using a recently developed system that initiates and elongates Ad templates in vitro. Replication depends on 5 purified components. Three are viral coded: the DNA polymerase (Pol), a precursor to the covalently-linked terminal protein (pTP) and the DNA binding protein (DBP). Two additional replication factors (I and II) are present in uninfected nuclear extracts. Factor II is a DNA topoisomerase. An understanding of the control of DNA replication is critical to an understanding of several important disease states including malignancies. Accordingly, this in vitro DNA replication system which is unique among eukaryocytes in that it can initiate new DNA strands will be used to study: (1) functionally active domains of the DNA Pol and pTP. Genes for these 2 viral coded proteins will be cloned and expressed in prokaryotic vectors. Site specific mutagenesis will allow the active domains of pTP and Pol to be mapped. (2) Temperature sensitive mutations in the Ad DBP, are correctable by second site mutations which require an RNA cofactor for full activity. The nature of the RNA will be studied. (3) Viral core proteins inhibit the in vitro DNA synthesis reaction and the mechanism of this inhibition will be studied. (4) The effect of several components (VP-16-213, VM26 and camptothecin), that inhibit Ad DNA synthesis by cleaving the DNA template can now be studied in vitro for all steps of the breakage reaction can be reproduced in such systems. (5) Studies will be continued on inhibition of Ad DNA elongation by antibody from patients with systemic lupus erythematosus. In addition to the inhibition already shown by sera with antibody to double-stranded DNA, antibodies to nuclear factors I and II will be specifically sought.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA011512-16
Application #
3163480
Study Section
Experimental Virology Study Section (EVR)
Project Start
1976-06-30
Project End
1989-04-30
Budget Start
1985-05-01
Budget End
1986-04-30
Support Year
16
Fiscal Year
1985
Total Cost
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Type
Schools of Medicine
DUNS #
009095365
City
Bronx
State
NY
Country
United States
Zip Code
10461
Brough, D E; Droguett, G; Horwitz, M S et al. (1993) Multiple functions of the adenovirus DNA-binding protein are required for efficient viral DNA synthesis. Virology 196:269-81
Joung, I; Horwitz, M S; Engler, J A (1991) Mutagenesis of conserved region I in the DNA polymerase from human adenovirus serotype 2. Virology 184:235-41
Fredman, J N; Pettit, S C; Horwitz, M S et al. (1991) Linker insertion mutations in the adenovirus preterminal protein that affect DNA replication activity in vivo and in vitro. J Virol 65:4591-7
Chen, M; Mermod, N; Horwitz, M S (1990) Protein-protein interactions between adenovirus DNA polymerase and nuclear factor I mediate formation of the DNA replication preinitiation complex. J Biol Chem 265:18634-42
Meyers, M L; Keating, K M; Roberts, W J et al. (1990) Purification and functional characterization of adenovirus ts111A DNA-binding protein. Fluorescence studies of protein-nucleic acid binding. J Biol Chem 265:5875-82
Chen, M; Horwitz, M S (1990) Replication of an adenovirus type 34 mutant DNA containing tandem reiterations of the inverted terminal repeat. Virology 179:567-75
Pettit, S C; Horwitz, M S; Engler, J A (1989) Mutations of the precursor to the terminal protein of adenovirus serotypes 2 and 5. J Virol 63:5244-50
Chen, M; Horwitz, M S (1989) Dissection of functional domains of adenovirus DNA polymerase by linker-insertion mutagenesis. Proc Natl Acad Sci U S A 86:6116-20
Pettit, S C; Horwitz, M S; Engler, J A (1988) Adenovirus preterminal protein synthesized in COS cells from cloned DNA is active in DNA replication in vitro. J Virol 62:496-500
Krevolin, M D; Horwitz, M S (1987) Functional interactions of the domains of the adenovirus DNA binding protein. Virology 156:167-70

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