The autonomous parvoviruses are small, single-stranded DNAcontaining viruses which can induce fetal demise and teratogenic effects during pregnancy and can interfere with both normal tissue turnover and the development of neoplastic disease in their adult host. The proposed research is aimed at understanding the molecular basis of replication of the autonomous parvoviral genome. MVM non-structural proteins with known replicative function, particularly the major nuclear phosphoprotein NS1, will be produced in eukaryotic and prokaryotic expression systems. The use of recombinant NS1 in binding and nicking assays with PCR-generated substrates will help to delineate the """"""""minimal origin"""""""" sequence within the viral terminal palindromes. A major focus of the proposed research will be to elucidate the mechanism of resolution of the palindromic viral termini from contatemeric replication intermediates. This process will be explored by studying the interaction of immunopurified recombinant NS proteins with linear duplex and hairpinned derivatives of the cloned viral junctions in vitro, particularly with those derived from the viral left-end:left-end fusion, which is resolved asymmetrically. The composition of the replication fork assembled on a single functional origin, and the structure of its product DNA, will be examined using specific inhibitors of DNA polymerases and accessory proteins. Functional studies on the NS proteins will be continued using a combination of biochemical ana genetic approaches. These efforts will be directed toward identifying the functional domains within the NS1 polypeptide responsible for DNA binding, for site-specific cleavage and DNA attachment, and for enzymatic activities such as ATPase and helicase.
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