Abstract 9315959 Carroll DNA molecules injected into Xenopus oocytes are subject to very efficient homologous recombination, providing that they are linear and have appropriately placed homologous sequences. This process has been demonstrated in nuclear extracts from oocytes, and it is known to depend on the action of a 5' - 3' exonuclease. It is proposed to purify this exonuclease from oocyte nuclei and to characterize its properties, particularly with respect to recombination. Partial amino acid sequences of the purified enzyme will be determined and antibodies that recognize the protein will be raised. cDNA clones representing the exonuclease mRNA will be isolated and sequenced, allowing comparisons with nuclease sequences from other organisms. Expression of the exonuclease from a cDNA clone will be engineered in bacteria (or in yeast) to allow isolation of larger quantities of the enzyme for enzymatic and immunological studies. Anti-nuclease antibodies will be used to determine where and when during development the enzyme is present. Particular attention will be paid to stages of oogenesis and early embryogenesis in which a role for the exonuclease in DNA metabolism has been hypothesized. Functions of the exonuclease will be examined directly by using antibodies that block its activity. The consequences of injecting such antibodies into fertilized eggs will be determined, with the expectation that defects in DNA replication and chromosome integrity may be found. Some effort will also be expended in identifying other activities that are necessary for recombination in oocytes. They will be sought by fractionation of nuclear extracts and assayed by reconstitution of recombination activity. Anticipated activities include DNA ligase, DNA polymerase, and a catalyst of the annealing of complementary single strands. *** Recombination events very like those demonstrated in Xenopus oocytes have been documented in cells of other organisms, including mammal ian cells. Thus, information of the oocyte exonuclease and other recombination proteins which should result from this work will have wide applicability, perhaps even in approaches to targeted gene manipulation. In any case, we will be more informed as to the critical parameters of homologous recombination in Xenopus oocytes. %%%
