Action of mung bean nuclease on supercoiled PM2 DNA.

D Kowalski,J P Sanford

doi:10.1016/s0021-9258(18)34455-7

Abstract

Single strand specific mung bean nuclease was used to probe for regions of altered secondary structure in supercoiled PM2 DNA. Supercoiled DNA is cleaved greater than or equal to 10,000 times faster than the relaxed topoisomer. Catalytic quantities of enzyme convert supercoiled DNA to both nicked-circular and unit length linear forms at pH 5 but to predominantly the nicked-circular form near neutral pH. At the elevated enzyme concentrations required to cleave relaxed DNA, unit length linear DNA and smaller fragments are produced from pH 5 to 7. One nick per supercoiled DNA molecule is introduced at pH 6.6. The nicks are repairable by DNA ligase and are not strand-specific. Snake venom phosphodiesterase selectively cleaves the strand opposite the nicks, permitting restriction endonuclease mapping. The nicks occur at three specific sites. Sites at 0.75 and 0.76 map units are cleaved with equal frequency, while a site at 0.82 is cleaved less frequently. The former sites map near one of the eight 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 and separation in supercoiled PM2 DNA.

Highlights

~ 1 0, 0 0 0times fasterthan the relaxed topoisomer
Transient, localized unwinding of duplex DNA structure may result in susceptible sites both within [2, 5, 6] and atthe ends ( 7 ) of a linear molecule. Hydrolysis at these transiently single-stranded regions is slow relative to that of single-stranded DNA and is highly dependent on reaction conditions which affect the secondary structure of DNA (2, 5, 7 ) .Model DNA heteroduplexes whose mismatched bases may be accommodated in a stacked helical structure are cleaved at extremely lowefficiencywhenonlyonebase is mismatched and at greater efficiency as the number of adjacent mismatched bases is increased [8]
The activity is optimal at acidic pH [7], cleavage of supercoiled DNA is readily measurable at neutral and alkaline pH values at elevated enzyme concentrations

Summary

Introduction

~ 1 0 , 0 0 0times fasterthan the relaxed topoisomer. Catalytic quantitiesof enzyme convert supercoileDdNA t o both nicked-circularand unit length linear forms at pH 5 but to predominantly the nicked-circular form near neutral pH. Transient, localized unwinding of duplex DNA structure may result in susceptible sites both within [2, 5, 6] and atthe ends ( 7 ) of a linear molecule. Hydrolysis at these transiently single-stranded regions is slow relative to that of single-stranded DNA and is highly dependent on reaction conditions which affect the secondary structure of DNA (2, 5, 7 ) .Model DNA heteroduplexes whose mismatched bases may be accommodated in a stacked helical structure are cleaved at extremely lowefficiencywhenonlyonebase is mismatched and at greater efficiency as the number of adjacent mismatched bases is increased [8].

Methods

Results

Conclusion