Cleavage of Circular, Superhelical Simian Virus 40 DNA to a Linear Duplex by S ₁ Nuclease

Abstract

S, nuclease, the single-strand specific nuclease from Aspergillus oryzae can cleave both strands of circular covalently closed, superhelical simian virus 40 (SV40) DNA to generate unit length linear duplex molecules with intact single strands. But circular, covalently closed, nonsuperhelical DNA, as well as linear duplex molecules, are relatively resistant to attack by the enzyme. These findings indicate that unpaired or weakly hydrogen-bonded regions, sensitive to the single strand-specific nuclease, occur or can be induced in superhelical DNA. Nicked, circular SV40 DNA can be cleaved on the opposite strand at or near the nick to yield linear molecules. S, nuclease may be a useful reagent for cleaving DNAs at regions containing single-strand nicks. Unlike the restriction endonucleases, S, nuclease probably does not cleave SV40 DNA at a specific nucleotide sequence. Rather, the sites of cleavage occur within regions that are readily denaturable in a topologically constrained superhelical molecule. At moderate salt concentrations (75 mM) SV40 DNA is cleaved once, most often within either one of the two following regions: the segments defined as 0.15 to 0.25 and 0.45 to 0.55 SV40 fractional length, clockwise, from the EcoR, restriction endonuclease cleavage site (defined as the zero position on the SV40 DNA map). In higher salt (250 mM) cleavage occurs preferentially within the 0.45 to 0.55 segment of the map.

Specific hydrolysis of rabbit globin messenger RNA by S1 nuclease

“…The influence of salt concentration and temperature on the SI digestion is consistent with changes in helical content in globin mRNA observed using optical methods (25,27), and is entirely analogous to results obtained with single strand nuclease digestions of SV40 DNA (14,15), MS2 RNA (13) and tRNA (17,34). The data reported here, along with our previous observations (25), show that agents known to disrupt helical structure (high temperature, low salt, formaldehyde) increase SI susceptibility.…”

“…S1 nuclease preferentially degrades single stranded regions in nucleic acids (11) and appears to have no base specificity (12,13). The enzyme has been widely used as a probe for single stranded structure in both DNA and RNA (14)(15)(16)(17)(18). The results presented here show that globin mRNA is 65-75% resistant to S1 nuclease and that susceptibility to enzymatic digestion changes with conditions known to affect helix stability.…”

Molecular characteristics and physical state of human papilloma virus DNA change with progressing malignancy: Studies in a patient with epidermodysplasia verruciformis

“…Nuclease S1 (Sl) converts form4 (closed circular) DNA into form I1 (open circular), and then into form 111 (linear): the reaction is rapid in the first step and much slower in the second step, while form-I11 DNA is rather resistant to S1 (Beard et al, 1973). When the DNA of the primary carcinoma was digested with 10 units of S1 (Fig.…”

Energy-structure correlations of plasmid DNA in different topological forms

“…The topological state of DNA affects its replication (2-4), recombination (5)(6)(7)(8), transcription (9)(10)(11), nucleosome assembly (12)(13)(14), and repair (15,16). The unique structural features, which characterize naturally occurring supercoiled molecules and determine their increased biological reactivity are single strand regions (17)(18)(19), cruciform structures (2,(20)(21)(22)(23)(24)(25) and stretches of left handed Z-form (26)(27)(28)(29)(30).…”