Association of poly(adenosine diphosphoribose) synthesis with DNA damage and repair in normal human lymphocytes.

A permeable cell technique was used to measure the alterations in synthesis of DNA and poly-(adenosine diphosphoribose) in normal human lymphocytes after treatment of the cells with different types of DNA-damaging agents. The lymphocytes showed an abrupt increase in the unscheduled synthesis of DNA and poly(adenosine diphosphoribose) in response to ultraviolet (UV) irradiation. The increases were apparent within 1 h and reached a maximum between 2 and 4 h after irradiation. The magnitude of the increases in DNA and poly(adenosine diphosphoribose) synthesis was dependent upon the UV dose. Alkaline CsCl gradient studies, with bromodeoxyuridine triphosphate density labeling of DNA, demonstrated that the unscheduled DNA synthesis, which occurred in response to UV irradiation, was actually a result of the repair mode of DNA synthesis. Similar increases in DNA synthesis, and poly(adenosine diphosphoribose) synthesis occurred when lymphocytes were treated with several other DNA-damaging agents, including bleomycin, N-methyl-N'-nitro-N-nitrosoguanidine or N-acetoxyacetyl aminofluorene. Treatment of lymphocytes with DNase, under conditions which allowed degradation of cellular DNA, also resulted in increased synthesis of poly(adenosine diphosphoribose). Cycloheximide did not inhibit the increase in synthesis of DNA or poly(adenosine diphosphoribose) that occurred in response to treatment with the DNA-damaging agents.

Synthesis of DNA and poly(adenosine diphosphate ribose) in normal and chronic lymphocytic leukemia lymphocytes.

Peripheral blood lymphocytes were isolated from 9 patients with chronic lymphocytic leukemia (CLL) and 12 normal control donors. The cells were assayed for synthesis of DNA and poly-(adenosine diphosphate ribose) (poly[ADPR]) immediately after isolation and on successive days following their treatment with phytohemagglutinin (PHA). Two different techniques were used to measure DNA synthesis. In the standard technique, DNA synthesis was measured by incubating intact cells with [(3)H]deoxythymidine. In the new technique, the lymphocytes were first rendered permeable to nucleotides, then DNA synthesis was measured by incubating them with [(3)H]deoxythymidine triphosphate in the presence of deoxyATP, deoxyGTP, deoxyCTP, ATP, and Mg(++). Both assays showed the anticipated rise in DNA synthesis after PHA stimulation of normal cells. PHA-stimulated lymphocytes from patients with CLL demonstrated low levels of DNA synthesis in both assay systems. The initial levels of poly(ADPR) synthesis were greater in CLL lymphocytes than in normal cells. Studies with a T-cell-depleted population of normal cells showed the same activity for poly(ADPR) synthesis that was demonstrated by the original population of normal cells. PHA stimulation produced an increase in poly(ADPR) synthesis in both the normal and CLL cells. The increase in poly(ADPR) synthesis in normal cells was coincident with the increase in DNA synthesis. The increase in poly(ADPR) synthesis in the CLL cells was dissociated from the delayed and diminished increase in DNA synthesis. Thus, CLL cells have higher than normal initial levels of poly(ADPR) synthesis. Poly(ADPR) synthesis is dissociated from DNA synthesis in CLL cells whereas it varies directly with DNA synthesis in normal lymphocytes.

ATP-independent DNA synthesis in vaccinia-infected L cells.

Mouse L cells can be made permeable to exogenous nucleotides by a cold shock in 0.01 M Tris . HCl pH 7.8, 0.25 M sucrose, 1 mM EDTA, 30 mM 2-mercaptoethanol and 4 mM MgCl2. DNA synthesis in permeabilized L cells requires ATP whereas DNA synthesis in permeabilized L cells that are infected with Vaccinia virus is ATP-independent. Permeabilized L cells that are infected with ultraviolet-irradiated virus show a marked suppression of DNA synthesis which is not corrected by an excess of deoxynucleoside triphosphates and ATP. The ATP-dependent and ATP-independent processes of DNA synthesis are inhibited to the same extent by Mal-Net, pHMB, ara CTP and phosphonoacetate. Concentrations of daunorubicin and cytembena, which cause marked inhibition of the ATP-dependent enzymes, only cause partial inhibition of the ATP-independent enzymes.

Poly(adenosine diphosphoribose) synthesis in ultraviolet-irradiated xeroderma pigmentosum cells reconstituted with Micrococcus luteus UV endonuclease.

Synthesis of DNA and poly(adenosine diphosphoribose) [poly(ADPR)] was examined in permeabilized xeroderma pigmentosum lymphoblasts (XP3BE) before and after UV irradiation and in the presence and absence of Micrococcus luteus UV endonuclease. M. luteus UV endonuclease had no effect on the level of DNA or poly(ADPR) synthesis in control, unirradiated cells. UV irradiation caused a decrease in replicative DNA synthesis without any significant change in poly(ADPR) synthesis. In UV-irradiated cells treated with M. luteus UV endonuclease, DNA synthesis was restored to a level slightly greater than in the unirradiated control cells, and poly(ADPR) synthesis increased by 2- to 4-fold. Time--course studies showed that the UV endonuclease dependent poly(ADPR) synthesis preceded the endonuclease-dependent DNA synthesis. Inhibition of endonuclease-dependent poly(ADPR) synthesis with 3-aminobenzamide, 5-methylnicotinamide, or theophylline produced a partial inhibition of the endonuclease-dependent DNA synthesis. Conversely, inhibition of the endonuclease-dependent DNA synthesis with dideoxythymidine triphosphate, phosphonoacetic acid, or aphidicolin had no effect on the endonuclease-dependent poly(ADPR) synthesis. These studies show that stimulation of poly(ADPR) synthesis in UV-irradiated cells occurs subsequent to the DNA strand breaks created by the specific action of the UV endonuclease on UV-irradiated DNA. The effect of the inhibitors of poly(ADPR) synthesis in UV-irradiated cells indicates that the endonuclease-stimulated DNA synthesis is dependent in part on the prior synthesis of poly(ADPR).