A system of endonucleases has been discovered in a marine Pseudomonas strain which contains a very highly single-strand specific activity as well as an activity which breaks a DNA chain everywhere a single-strand scission occurs, resulting in duplex fragments which possess short single-standed ends. Highly supercoiled closed circular DNA is attacked by these nucleases to yield full length linear duplex molecules with cohesive ends; these can be joined with polynucleotide ligase to make oligomeric molecules. As dimeric species resulting from joining two identical linear molecules are found ("head-to-head" or "tail-to-tail" joining), the sequences cleaved must be of the antisymmetric palindromic (duplicated inverted) variety. Fully duplex linear DNA's, or closed circular DNA's with few superhelical turns, are not attacked. The attack is not at a unique site in the closed circular molecule. The activities are not significantly repressed at ionic strengths up to 3 M. It is proposed to purify and characterize the system of nucleases. It is of particular interest to ascertain whether the single strand-specific and nick-specific activities are separable. The activity of the system against various nucleic acid substrates and under various conditions is readily monitored in the analytical ultracentrifuge. Experiments are proposed with the purified system as to the metal ion cofactor requirements, behavior at elevated ionic strengths and temperatures, DNA and RNA substrate specificity, degree of supercoiling in closed circular DNA required for activity, and the nature of the single-stranded ends resulting from cleavage with the nick-specific activity. The possibility that the enzyme system, used in conjunction with polynucleotide ligase, may provide a new method for joining together DNA's from different viruses and episomes will be tested. The use of the system for the controlled molecular weight degradation of double-stranded nucleic acids without nicks in the duplex fragments will be explored, as well as its use as an improved system for isolating duplex regions from heteroduplex nucleic acid molecules.