We propose to further study the structure and function of the avian retrovirus polymerase gene products. Among the structural studies, the DNA polymerase and RNase H active sites will be ordered on the alpha polypeptide, the alphabeta DNA polymerase will be cleaved by p15 to generate an in vitro product similar to the in vivo pp32 DNA endonuclease and the role of phosphorylation in pol polyprotein processing and pol associated activities will be studied. We will futher develop our initial finding that the in vivo pp32 protein forms a nucleosome-like structure with selected regions on retrovirus long terminal repeat (LTR) DNA. The cloned LTR DNA will be resected to define the functional binding limits of the pp32 protein. The DNA nickase activity associated with pp32 will be further characterized with respect to its possible role in viral DNA integration and efforts will be extended to define the relationship of the pp32 DNA nickase activity with its associated DNA binding proterties. A major effort will be to develop various in vivo systems to enhance our biological understanding of the pol DNA endonuclease function(s). We will construct functional virus mutants whose polymerase possesses only a temperature-sensitive endonuclease function with normal DNA polymerase and RNase H active sites. Current techniques in site-directed mutagenesis of DNA will be utilized to induce single base changes in the retrovirus pol gene region of cloned retrovirus DNA coding for the DNA endonuclease site. These mutants will hopefully provide pertinent data for defining the biological role of pp32 and also complement our biochemical studies of pp32 and alphabeta DNA polymerase.
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