Reverse transcription plays a key role in virus replication and quite possibly in other genomoic arrangements, hence it is desirable to fully understand it. One of the enzymes involved is DNA endonuclease (pp32). Dr. Grandgenett has found that pp32 phosphorylated and he now hopes to locate the phosphorylated sites. Labeling studies indicate that the three serines in the COOH-terminal one-third of pp32 are phosphorylated. The exact location of the serine will be established and the biological significance of each phosphorylated site defined. The biological significance of this phosphorylation will be assessed by generation of modified p-SER sites by oligonucleotide mutagenesis of infectious cloned Prague A DNA. This should permit the alignment of phosphorylation to a defect at the molecular level. Attempts will be made to define what biological properties are associated with the pp32 expressed in cells by several eucaryotic vectors constructed under the previous grant. Since the amino acid sequence of pp32 is highly conserved in pol of certain eucaryotic transposons, defining the properties of pp32 intracellularly may be beneficial in understanding these mobile genetic units. The investigator is one of the few individuals working in this area using a biochemical approach and thus this project merits a high priority for funding.
