The 12/23 rule is a critical step for regulation of V(D)J recombination. To date, only the RAG proteins and high mobility group protein 1 or 2 have been implicated in 12/23 regulation. Through protein fractionation and biochemical experiments, we find that Ku70/Ku80 and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) modulate RAG-mediated cleavage. Modulation of cleavage by Ku70/80 an...

Restriction endonucleases recognize short DNA sequences and cleave double-stranded DNA at specific sites within or adjacent to the recognition sequences. Restriction endonuclease cleavage of DNA into discrete fragments is one of the most basic procedures in molecular biology. The first method presented in this unit is the cleavage of a single DNA sample with a single restriction endonuclease. A...

The utility of restriction endonucleases as a tool in molecular biology is in large part due to the high degree of specificity with which they cleave well-characterized DNA recognition sequences. The specificity of restriction endonucleases is not absolute, yet many commonly used assays of biological phenomena and contemporary molecular biology techniques rely on the premise that restriction en...

The natural product jadomycin B, isolated from Streptomyces venezeulae ISP5230, has been found to cleave DNA in the presence of Cu(II) ions without the requirement for an external reducing agent. The efficiency of DNA cleavage was probed using supercoiled plasmid DNA in buffered solution as a model environment. EC₅₀ and t(½) values for cleavage were 1.7 μM and 0.75 h, respectively, and varied ±...

The cytotoxicity of several important antitumor drugs depends on formation of the covalent topoisomerase-DNA cleavage complex. However, cellular processes such as DNA replication are necessary to convert the cleavage complex into a cytotoxic lesion, but the molecular mechanism of this conversion and the precise nature of the cytotoxic lesion are unknown. Using a bacteriophage T4 model system, w...

Antineoplastic intercalating agents such as 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) stabilize a cleavable com plex between topoisomerase II and DNA. The production of proteinassociated DNA cleavage in whole cells exposed to m-AMSA is thought to represent the cellular correlate of this topoisomerase II-mediated reaction. Protein-associated DNA cleavage can be quantified in mam ma...

Antineoplastic intercalating agents such as 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) stabilize a cleavable complex between topoisomerase II and DNA. The production of protein-associated DNA cleavage in whole cells exposed to m-AMSA is thought to represent the cellular correlate of this topoisomerase II-mediated reaction. Protein-associated DNA cleavage can be quantified in mammal...

Sister chromatid separation is triggered by the separase-catalyzed cleavage of cohesin. This process is temporally controlled by cell-cycle-dependent factors, but its biochemical mechanism and spatial regulation remain poorly understood. We report that cohesin cleavage by human separase requires DNA in a sequence-nonspecific manner. Separase binds to DNA in vitro, but its proteolytic activity, ...

Mycobacterium tuberculosis encodes only a single type II topoisomerase, gyrase. As a result, this enzyme likely carries out the cellular functions normally performed by canonical gyrase and topoisomerase IV, both in front of and behind the replication fork. In addition, it is the sole target for quinolone antibacterials in this species. Because quinolone-induced DNA strand breaks generated on p...

A cleavage-deficient variant of NotI restriction endonuclease (GCGGCCGC) was isolated by random mutagenesis of the notIR gene. The NotI variant D160N was shown to bind DNA and protect plasmid DNA from EagI (CGGCCG) and NotI digestions. The EDTA-resistant BmrI restriction endonuclease cleaves DNA sequence ACTGGG N5/N4. The N-terminal cleavage domain of BmrI (residues 1-198) with non-specific nuc...