No abstract

T7 endonuclease I is known to bind and cleave four-way junctions in DNA. Since these junctions serve as analogues of Holliday junctions that arise during genetic recombination, we have investigated the action of T7 endonuclease I on recombination intermediates containing Holliday junctions. We find that addition of T7 endonuclease I to strand exchange reactions catalysed by RecA protein of Escherichia coli leads to the formation of duplex products that correspond to 'patch' and 'splice' type recombinants. Resolution of the recombination intermediates occurs by the introduction of nicks at the site of the Holliday junction. The recombinant molecules contain 5'-phosphate and 3'-hydroxyl termini which may be ligated to restore the integrity of the DNA.

Preferential cleavage of Saccharomyces cerevisiae chromosomes in internucleosomal (linker) regions and nonspecific degradation of chromatin by an anticancer antibiotic which degrades DNA were investigated and found to increase in consecutive stages of growth. Cleavage of DNA in internucleosomal regions and intensities and multiplicities of nucleosomal bands were dependent on drug concentration, growth phase of the cells, and length of incubation. Cellular DNA was least degraded during logarithmic phase. After cells progressed only one generation in logarithmic phase, low concentrations (6.7 x 10-7 to 3.4 x 10-6 M) of bleomycin produced approximately three to seven times more DNA breaks. Internucleosomal cleavage was highest, and the most extended oligonucleosomal series and extensive chromatin degradation were observed during stationary phase. It is concluded that the growth phase of cells is critical in determining amounts of the highly preferential cleavage in internucleosomal regions and overall breakage and degradation of DNA. Mononucleosomal bands were most intense, indicating the greatest accumulation of DNA of this size. Mean mononucleosomal lengths were 165.9 + 3.9 base pairs, in agreement with yeast mononucleosomal lengths. As high-molecular-weight chromatin was digested by bleomycin, oligonucleosomes and, eventually, mononucleosomes became digested. Therefore, it is also concluded that bleomycin degradation of oligonucleosomes and trimming of DNA linker regions proceed to degradation of the monosomes (core plus linker DNA).We consider in the current investigation that the growth phase of cells could be a crucial factor in determining lesions produced in cellular chromatin. The chromatin conformation characteristic for chromosomal organization in eucaryotic cells certainly can be very important in determining the nature and extent of chemical reactivity with genomic structures. For example, DNA is preferentially cleaved in internucleosomal (linker) regions of chromatin in isolated mammalian nuclei (18,22,23), in intact human K562 cells (46), and in Saccharomyces cerevisiae (40) by the unique chemical action of the low-molecular-weight glycopeptides (Mr, approximately 1,500 to 1,600 [ Fig. 1]) in the bleomycin family of anticancer antibiotics (4,12,14,20,21,30,31,39,47,59,60,62). Bleomycins form single-and double-strand breaks in DNA (e.g., see references 2, 7, 15, 16, 29, 39, 41, 44, and 60 sensitive to killing by bleomycin in plateau phase than when growing exponentially, but the reverse was true for mouse tumor cells in an earlier study (71) and for Chinese hamster V79 (28), Burkitt's lymphoma (32,66), and human colon carcinoma (8) (10,36,39,44,51) from starting inocula of 8 x 106 cells per ml. Cells were harvested by centrifugation at 4°C in a Dupont Sorvall RC-SB SS34 rotor at 2,500 x g and washed twice with deionized water.