The overall objectives of the proposal are to investigate the molecular mechanism of DNA replication and the evolution of genes essential for transformation in adenoviruses. The unique property of inverted terminal repetitions (ITR) common to all adenoviruses seems to play an unknown role in DNA replication. We have focused our effort in the past to determine the nucleotide sequences of this region from representative members of group A (highly oncogenic), group B (weakly oncogenic), group C and E (non-oncogenic) adenoviruses. The length and DNA sequence of this region diverge considerably except for a unique region of homology present at an exactly identical location near the termini. Analysis of the intermediates of replication of adenovirus types 2 & 5 from the injected cells or from the in vitro replication systems have shown, from the work cone in several laboratories, that initiation of DNA replication takes places at or near either end of the viral DNA. Displacement of one of the parental strands takes place as the daughter strand synthesis proceeds in the 5' to 3' direction. It is not clear how the displaced strand replicates. A model has been proposed that it replicates via a circular """"""""panhandle"""""""" intermediate formed due to the presence of ITR. I propose to study the role of ITR in DNA replication of displaced parental strands using DNA cross-linking psoralens. The role of the unique 14 bp sequence which might function as a recognition signal for initiation of DNA replication will be studied by using chemically synthesized oligodeoxynucleotides as site-specific mutagens as well as by misincorporation of excisi-on-resistant analogues of deoxynucleoside triphosphates. Transitions, transversions and deletion mutants will be generated and assayed for DNA replication in vivo and in vitro. Our preliminary studies indicated that 9 amino acridine inhibits adenovirus DNA replication in vivo. We will pursue this aspect further to understand the molecular mechanism of this inhibition.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA033099-03
Application #
3171045
Study Section
Experimental Virology Study Section (EVR)
Project Start
1983-12-01
Project End
1987-11-30
Budget Start
1985-12-01
Budget End
1987-11-30
Support Year
3
Fiscal Year
1986
Total Cost
Indirect Cost
Name
University of Kansas
Department
Type
Schools of Medicine
DUNS #
016060860
City
Kansas City
State
KS
Country
United States
Zip Code
66160
Chen, H; Padmanabhan, R (1997) A simplified method of screening for isolation of recombination vaccinia virus. Methods Mol Biol 62:199-206
Chen, H; Campisi, J; Padmanabhan, R (1996) SV40 large T antigen transactivates the human cdc2 promoter by inducing a CCAAT box binding factor. J Biol Chem 271:13959-67
Ramachandra, M; Sasaguri, Y; Nakano, R et al. (1996) Heterologous expression, purification, and characterization of adenovirus DNA polymerase and preterminal protein. Methods Enzymol 275:168-94
Ramachandra, M; Padmanabhan, R (1995) Expression, nuclear transport, and phosphorylation of adenovirus DNA replication proteins. Curr Top Microbiol Immunol 199 ( Pt 2):50-88
Chen, H; Padmanabhan, R (1994) A modified method for isolation of recombinant vaccinia virus. Biotechniques 17:40, 42
Kusukawa, J; Ramachandra, M; Nakano, R et al. (1994) Phosphorylation-dependent interaction of adenovirus preterminal protein with the viral origin of DNA replication. J Biol Chem 269:2189-96
Chen, H; Ramachandra, M; Padmanabhan, R (1994) Biochemical characterization of a temperature-sensitive adenovirus DNA polymerase. Virology 205:364-70
Ramachandra, M; Padmanabhan, R (1993) Adenovirus DNA polymerase is phosphorylated by a stably associated histone H1 kinase. J Biol Chem 268:17448-56
Ramachandra, M; Nakano, R; Mohan, P M et al. (1993) Adenovirus DNA polymerase is a phosphoprotein. J Biol Chem 268:442-8
Nakano, R; Zhao, L J; Padmanabhan, R (1991) Overproduction of adenovirus DNA polymerase and preterminal protein in HeLa cells. Gene 105:173-8

Showing the most recent 10 out of 15 publications