A Comparative Study For Supercoiled Pm2 Dna In Which Action Performed By Mung Bean Nuclease |

ABSTRACT:

Single strand specific mung bean nuclease was used toprobe for regions of altered secondary structure in supercoiled PM2 DNA.Supercoiled DNA is cleaved >10,000 times faster than the relaxed topoisomer.Catalytic quantities of enzyme convert supercoiled DNA to both nicked-circularand unit length linear forms at pH 5 but to predominantly the nicked-circularform near neutral pH. At the elevated enzyme concentrations required to cleaverelaxed DNA, unit length linear DNA and smaller fragments are produced from pH5 to 7. One nick per supercoiled DNA molecule is introduced at pH6.6. The nicks are repairable by DNA ligase and are not strand-specific. Snakevenom phosphodiesterase selectively cleaves the strand opposite the nicks,permitting restriction endonuclease mapping. The nicks occur at three specificsites. Sites at 0.75 and 0.76 map units are cleaved with equal frequency, whilea site at 0.82 is cleaved less frequently. The former sites map near one of theeight known early denaturation regions of PM2 DNA, while the latter does not.Since most early denaturation sites are not cleaved, sites other than these dA+ dT-rich regions may be the preferred locations of strand unwinding andseparation in supercoiled PM2 DNA. It is shown that local denaturation can be a naturalconsequence of supercoiling, even in environments where base pairing of linearDNA is energetically favored. Any change in the molecular total twist from itsunstressed value is partitioned between local denaturation and smooth twistingin both the native and coil regions so as to minimize the total conformationalfree energy involved. Threshold degrees of torsional deformation are found forthe existence of stable, locally melted conformations. As these thresholds aresurpassed, the number of denatured bases increase smoothly from zero. Existing experimentalevidence regarding denaturation in supercoiled DNA is in good agreement withthe predictions of this theory. In addition, from existing data one canestimate the partitioning of superhelicity between twisting and writhing.Possible consequences of stress-induced strand separation on the accessibilityof the DNA to enzyme attack are discussed. Control of local melting by DNAtopoisomerases and DNA gyrases could regulate diverse events involved intranscription, replication, recombination, and repair.