Presence Of Single-strand Breaks Research Articles

Role of Histone Tails and Single Strand DNA Breaks in Nucleosomal Arrest of RNA Polymerase.

Transcription through nucleosomes by RNA polymerases (RNAP) is accompanied by formation of small intranucleosomal DNA loops (i-loops). The i-loops form more efficiently in the presence of single-strand breaks or gaps in a non-template DNA strand (NT-SSBs) and induce arrest of transcribing RNAP, thus allowing detection of NT-SSBs by the enzyme. Here we examined the role of histone tails and extranucleosomal NT-SSBs in i-loop formation and arrest of RNAP during transcription of promoter-proximal region of nucleosomal DNA. NT-SSBs present in linker DNA induce arrest of RNAP +1 to +15 bp in the nucleosome, suggesting formation of the i-loops; the arrest is more efficient in the presence of the histone tails. Consistently, DNA footprinting reveals formation of an i-loop after stalling RNAP at the position +2 and backtracking to position +1. The data suggest that histone tails and NT-SSBs present in linker DNA strongly facilitate formation of the i-loops during transcription through the promoter-proximal region of nucleosomal DNA.

DNA damaging capability of hematoporphyrin towards DNAs of various accessibilities

In this work we wanted to verify that photoactivation of DNA-non-binding porphyrin derivative hematoporphyrin IX (Hp) is able to induce damages in DNAs of various accessibilities such as B-conformation and superhelical isolated DNA, nucleoprotein complex and intracellular DNAs. It was found that photodynamic reaction of Hp results significant changes in thermal stability of isolated T7 DNA and induces single strand breaks in supercoiled Bluescript plasmid isolated from Escherichia coli cells. As optical melting measurements revealed, the irradiation of photosensitized T7 nucleoprotein complex leads to a destabilization of the protein capsid. The photodynamic reaction affected both the protein structure and DNA–protein interaction, however, the parameters corresponding to the DNA denaturation are not influenced. The accumulation of Hp in HeLa cells was followed by laser scanning confocal microscopy. The picture received is typical for lipophilic dyes. When Hp loaded cells were irradiated, a reduction of viability could be observed in a concentration and a light dose dependent manner; 12 μM porphyrin induced almost complete cell killing after 30 min irradiation. After similar treatment, alkaline agarose gel electrophoresis of isolated nuclear DNA did not show the presence of single strand breaks. The alkaline comet assay also failed to demonstrate any DNA damage in HeLa cells. We also considered the possibility of the generation of damages in intracellular SV40 DNA. According to the electropherograms there was no difference between the patterns of DNAs from treated and control samples.

A CAF-1-PCNA-mediated chromatin assembly pathway triggered by sensing DNA damage.

Sensing DNA damage is crucial for the maintenance of genomic integrity and cell cycle progression. The participation of chromatin in these events is becoming of increasing interest. We show that the presence of single-strand breaks and gaps, formed either directly or during DNA damage processing, can trigger the propagation of nucleosomal arrays. This nucleosome assembly pathway involves the histone chaperone chromatin assembly factor 1 (CAF-1). The largest subunit (p150) of this factor interacts directly with proliferating cell nuclear antigen (PCNA), and critical regions for this interaction on both proteins have been mapped. To isolate proteins specifically recruited during DNA repair, damaged DNA linked to magnetic beads was used. The binding of both PCNA and CAF-1 to this damaged DNA was dependent on the number of DNA lesions and required ATP. Chromatin assembly linked to the repair of single-strand breaks was disrupted by depletion of PCNA from a cell-free system. This defect was rescued by complementation with recombinant PCNA, arguing for role of PCNA in mediating chromatin assembly linked to DNA repair. We discuss the importance of the PCNA-CAF-1 interaction in the context of DNA damage processing and checkpoint control.

ADP-ribosyltransferase activity during the friend virus-induced murine erythroleukemia cell differentiation.

A variety of chemical agents that are known to induce erythrodifferentiation in the Friend virus-induced murine erythroleukemia (MEL) cell have been suggested to mediate DNA cleavage in cultured cells prior to differentiation. The activation of the nuclear enzyme, ADP-ribosyltransferase, depends upon the presence of single strand breaks in DNA. If dimethyl sulfoxide (Me2SO) causes DNA breakage, it would be expected that the activity of ADP-ribosyltransferase would increase. A study of ADP-ribosyltransferase activity during cell growth indicates that both Me2SO-treated and untreated MEL cells exhibit a similar increase in the enzyme activity but the increase in Me2SO-treated cells is delayed by a few hours. When examined at comparable stages of growth, both treated and untreated cells show almost identical levels of enzyme activity. The present data thus do not support the contention that Me2SO induces DNA breakage in the MEL cells.

Differential DNA Damage Induced by Chemical Mutagens in Cells Growing in a Modified Selye’s Granuloma Pouch

Rapid growth of granulation tissue was induced in rats by injection of croton oil into a subcutaneous air pocket. Growth characteristics of the granulation tissue were evaluated by histopathologic techniques and measurement of 3H-thymidine incorporation into DNA. The DNA of cells growing in the granuloma was labeled with 3H-thymidine. Subsequently, 4 classes of test chemicals (monofunctional and polyfunctional alkylating agents, DNA intercalating agents and chemicals not known to interact with DNA) were injected intraperitoneally. The presence of single-strand breaks was assayed in DNA of granuloma tissue using the alkaline elution technique. DNA breaks were primarily induced by monofunctional alkylating agents and were characteristic for each compound. DNA from animals treated with polyfunctional alkylating agents and DNA-intercalating agents showed a variable degree of resistance to methylmethane sulfonate-induced DNA breakage.

KINETIC STUDIES ON THE THERMAL DENATURATION OF DEOXYRIBONUCLEIC ACID WITH A HIGH-FREQUENCY HEATING METHOD.

The rate of heat denaturation of calf-thymus, salmon-sperm and bacterial DNA's was studied with a high-frequency electric heating system. It was found that the rate of heat denaturation of DNA is rather slow, requiring more than 3 sec. The rate of denaturation is dependent on the guanine-cytosine content, and it is faster when the guanine-cytosine content is smaller. The rates are also faster below pH 5.0. Furthermore, the rate is faster in the presence of 8 M urea in the case of salmon-sperm DNA but is not affected by urea in the case of calf-thymus DNA. The relationship between the rate of unwinding and the length of DNA is also investigated but because of the possible presence of single-strand breaks, the results are complicated and no conclusion is reached concerning this problem.