Discrete Fragments Produced by Limited Digestion of Superhelical PM2 DNA with Venom Phosphodiesterase

Limited digestion of superhelical (form I) PM2 DNA by venom phosphodiesterase yields unit length linear (form III) molecules plus a series of discrete fragments. The positions of eight of these fragments on the genome map were determined with the use of restriction endonucleases Hpa II and HindIII. These positions were compared with the locations of the eight early denaturation regions in form I PM2 DNA which have been determined previously (Brack, C., Bickle, T. A., and Yuan, R. (1975) J. Mol. Biol. 96,693-702). Five phosphodie&erase-susceptible sites were identified and four of these are at or near early denaturation regions but one is not. The fragments produced by phosphodiesterase can be paired so that the sum of each pairs’ molecular weights is that of intact PM2 DNA. Only one member of each pair is susceptible to Hpa II, known to cleave PM2 DNA at a single locus, and the two members of each pair are produced in equimolar amounts. Therefore, each fragment is generated by two nearly simultaneous cleavages of form I PM2 DNA. For each fragment, one cleavage occurs at 15% on the genome map, and the other at either 62%, 72%, 78% or 85%. Digestion at low M$+ concentration generates unit length form III but not fragmented DNA. The ratio of unit length to fragmented form III DNA is independent of enzyme concentration over a range of at least 50-fold. Models accounting for the enzymatic mechanism and the role of the tertiary structure of PM2 DNA are discussed.