DNA gyrase, in the presence of the inhibitor oxolinic acid, can induce double-strand DNA breakage at specific sites. The sequences at several sites have been determined. In addition, the structure of complexes formed between DNA gyrase and restriction fragments containing an oxolinic acid-promoted cleavage site has been examined by DNase protection methods. DNA gyrase protects more than 120 bas...

In order to study the sequence specificity of double-strand DNA cleavage by Drosophila topoisomerase II, we have mapped and sequenced 16 strong and 47 weak cleavage sites in the recombinant plasmid p pi 25.1. Analysis of the nucleotide and dinucleotide frequencies in the region near the site of phosphodiester bond breakage revealed a nonrandom distribution. The nucleotide frequencies observed w...

DNA minicircles found within the kinetoplast of the trypanosomatid Crithidia fasciculata, like those of most other kinetoplastid species, are heterogeneous in sequence. The pattern of minicircle DNA fragments generated by cleavage of kinetoplast DNA with various restriction enzymes has been used to demonstrate this heterogeneity. Here we describe a strain of Crithidia fasciculata in which more ...

Site-specific recombination systems are useful tools for chromosome engineering in vivo and site-specific DNA cleavage methods have applications in genome analysis and gene isolation. Here, we report a new method to fragment chromosomes in vitro using the Cre-lox site-specific recombination system. Two lox sites were targeted into the 5.7 Mb chromosomes I of Schizosaccharomyces pombe. In vitro ...

Simian virus 40 (SV40) early pre-mRNA is spliced using either of two alternative 5' splice sites and a common 3' splice site to produce two mRNAs that encode the T and t antigens. We have studied alternative splicing of SV40 early pre-mRNA in vitro using a HeLa cell nuclear extract. Synthetic SV40 early transcripts are processed to T and t antigen mRNAs in vitro. As in SV40-infected cells in vi...

The MvaI restriction endonuclease cuts 5'-CC↓AGG-3'/5'-CC↑TGG-3' sites as indicated by the arrows. N4-methylation of the inner cytosines (C(m4)CAGG/C(m4)CTGG) protects the site against MvaI cleavage. Here, we show that MvaI nicks the G-strand of the related sequence (CCGGG/CCCGG, BcnI site) if the inner cytosines are C5-methylated: C(m5)C↓GGG/CC(m5)CGG. At M.SssI-methylated SmaI sites, where tw...

Type II topoisomerases are the targets of several classes of chemotherapeutic agents that stabilize an intermediate of the catalytic cycle with the enzyme covalently linked to cleaved DNA. We have used 3-azido-AMSA [4'-(3-azido-9-acridinylamino)methanesulfon-m-anisidide], a photo-activatible analog of the inhibitor m-AMSA [4'-(9-acridinylamino)methanesulfon-m-anisidide], to localize the inhibit...

32P-End-labeled restriction fragments derived from pBR322 and pUC9 DNAs were reacted with D-glucosamine or D-glucosamine-6-phosphate in the presence of Cu2+, and, after being heated at 90 degrees C in aqueous piperidine, the DNA products were analyzed on polyacrylamide gels for the sequence-specificity of alkali-labile cleavaged sites. The intensity of oligonucleotide bands of cleavaged sites w...

BACKGROUND Type II transmembrane serine proteases (TTSPs) are a family of cell membrane tethered serine proteases with unclear roles as their cleavage site specificities and substrate degradomes have not been fully elucidated. Indeed just 52 cleavage sites are annotated in MEROPS, the database of proteases, their substrates and inhibitors. METHODOLOGY/PRINCIPAL FINDING To profile the active s...

Self-cleavage of both plus and minus RNA transcripts of the 247-residue avocado sunblotch viroid (ASBV), prepared from tandem dimeric cDNA clones, occurs specifically at two sites in each transcript to give monomeric plus and minus species. The cleavage reaction occurs both during transcription and on incubation of purified transcripts at pH 8 and 37 degrees C in the presence of magnesium ions ...