Tadahiko Ando

Estimation of the double-helical content in various single-stranded nucleic acids by treatment with a single strand-specific nuclease.

Abstract Single-stranded nucleic acids from various sources were digested at low temperature with nuclease S1-specific for single-stranded polymers. The order of S1-resistance was : (double-stranded DNA) > poly(A) · poly(U) ⪢ ribosomal RNA , transfer RNA, Qβ RNA, MS2 RNA, TMV RNA > f 1 DNA ⪢ heat-denatured DNA ⪢ poly(U) . In an experiment using intact f1 DNA and fragmented f1 DNA, the S1 resistance coincided well with the degree of hyperchromicity exhibited by the tested DNA. The S1 resistance was changeable depending on the reaction temperature and the ionic strength in the reaction mixture.

Cleavage of ultraviolet light-irradiated DNA by single strand-specific S1 endonuclease

Abstract It was evidenced that the single strand-specific S1 endonuclease could cleave the ultraviolet light-irradiated T7 DNA. The cleavage of ultraviolet light-irradiated T7 DNA by S1 endonuclease was studied by sucrose density gradient centrifugation. The extent of cleavage was proportional to the dose of ultraviolet light given, the concentration of endonuclease and the ionic strength in the reaction. The cleavage consisted of both single-strand and double-strand breaks. The double-strand breaks were observed even at relatively lower dose of ultraviolet light. It seems likely that S1 endonuclease can recognize the alteration in the double-helical structure produced by ultraviolet light-irradiation rather than specifically attack ultraviolet light-induced photoproducts.

Site-specific fragmentation of bacteriophage T5 DNA by single-strand-specific S1 endonuclease.

Abstract Bacteriophage T5+ DNA molecule has been known to contain single-strand interruptions at defined positions. From the sedimentation analysis of the digestion products of the DNA by single-strand-specific S1 endonuclease, it was shown that S1 can make cleavages on the opposite strand at the pre-existing interruptions to generate the fragmented DNA site-specifically under the appropriate condition.

Characterization of E.coli RNA polymerase-binding sites on fl RFI DNA

Summary E. coli RNA polymerase-binding sites on fl RFI DNA were isolated as DNA fraction (1.9% of total DNA) protected by RNA polymerase against the nucleolytic digestion. These polymerase-protected DNAs had the properties as follows:(1) They were not obtained in the absence of sigma factor. (2) They were short double-stranded DNA fragments(35–40 base pairs long) and enriched in A-T base pairs (65%). (3) After heatdenaturation, they exhibited the resistance to the single strandspecific nuclease to some extent, but the nuclease-resistance was very temperature dependent. They behave not like GC-rich regions on fl DNA, which have very stable hairpin-like structure.

Substrate specificity of the three endonucleases isolated from phage λC1-infected Escherichia coli

Abstract Three endonucleases isolated from phage λCI-infected Escherichia coli C600 were designated as λ endonucleases I, II and III. Whereas λ endonucleases I and II hydrolyzed phage λ DNA into acid-soluble nucleotides to a considerable extent, they liberated no significant amount of acid-soluble nucleotides from DNAs of E. coli, phage T4 and phage T7. These two enzymes differed from each other in their elution profiles from a hydroxylapatite column, pH optimum, and in their responce to metallic ions and a chelating agent. λ endonuclease III was a DNA-nicking enzyme. It introduced a 10-fold larger number of single-strand breaks in λ DNA than in E. coli DNA. No break was introduced in phage T4 DNA.