Vol. 121, No. 3 Printed in U.S.A.

JOURNAL OF BACTERIOLOGY, Mar. 1975, p. 1211-1213 Copyright 0 1975 American Society for Microbiology

Endonuclease from Dictyostelium discoideum That Attacks Ultraviolet-Irradiated Deoxyribonucleic Acid R. A. DEERING* AND D. S. JENSEN Biophysics Department, Pennsylvania State University, University Park, Pennsylvania 16802 Received for publication 21 November 1974

An endonuclease activity yielding single-strand breaks in ultraviolet lightirradiated OX-174 RF I DNA in vitro has been detected in homogenates of the amoebae of the cellular slime mold, Dictyostelium discoideum.

Endonucleases that produce single-strand breaks in deoxyribonucleic acid (DNA) damaged by radiation or chemicals have been detected in extracts from several types of cells (5). These are often associated with the initial steps in the enzymatic repair of damaged DNA in vivo (2, 5). In recent studies of the action of ultraviolet light (UV) on the cellular slime mold, Dictyostelium discoideum, the appearance of strand breaks in DNA in vivo following UV irradiation has suggested the presence of such an endonuclease(s) in this organism (A. Guialis, Ph.D. thesis, Pennsylvania State University, 1974; Guialis and Deering, manuscript in preparation). We report here the detection in vitro of an endonuclease activity from D. discoideum that produces single-strand breaks in UV-irradiated DNA. Endonuclease activity was measured by the conversion of supercoiled, double-strand XX-174 replicative form I (RF I) to RF II, the circular, double-strand form containing one or more single-strand breaks. RF I, labeled with [3H]thymidine and prepared by the method of Burton and Sinsheimer (1), was the gift of L. R. Burns of this department. The preparation as used was 95% RF I and 5% RF II, at 4 ytg/ml in 0.01 M tris(hydroxymethyl)aminomethane (Tris), 0.001 M ethylenediaminetetraacetate (EDTA), pH 8. For the assay, 30 ,ul of the RF preparation, either unirradiated or irradiated at 0 C with 254 nm UV light at 100 ergs/mm2 per s, was mixed with 15 ,l of cell extract (see below) and 30 ql of unirradiated calf thymus DNA (Worthington, Freehold, N.J.) at 2 mg/ml in 0.1 M Tris, 0.001 M EDTA, pH 7.0 (ultrasonically treated to reduce viscosity). The calf thymus DNA was required to compete for nonspecific endonucleases to reduce their interference with the detection of endonuclease active on UVirradiated DNA. The reaction proceeded at 23 C and was stopped by the addition of 40 Al of

8% sodium lauryl sarcosine. The sarcosine effectively stopped the reaction and did not interfere with the subsequent gradient sedimentation of the RF DNA. Neutral sucrose gradients were used for the analysis instead of alkaline gradients so that alkali-labile bonds would not be a factor in the results. The gradients were 5 to 18% sucrose in 0.01 M Tris, 0.001 M EDTA (pH. 8.0). The entire reaction mixture was layered on these gradients at room temperature and sedimented at 20 C for 100 min in a Beckman SW50.1 rotor at 50,000 rpm. Fractions of 6 drops each were collected, 0.1 ml of water and 2.0 ml of scintillation fluid were added (3), and the resulting emulsion was counted in a Beckman LS-250 scintillation counter. Under these conditions, RF I and RF II would be expected to sediment at s2O. w of 24 and 17, respectively (6). D. discoideium NC-4 amoeboid cells to be used for extracts were grown to stationary phase (107/ml) on Escherichia coli B/r at 1010/ml in phosphate-buffered salts (3, 4). These cells were washed free of most of the few unconsumed bacterial cells by repeated low speed differential centrifugations (175 x g for 3 min). The amoebae were then incubated at 2 x 107/ml in fresh phosphate-buffered saline with no bacteria for 17 to 18 h to remove metabolically any traces of ingested bacterial components (see reference 3). They were then again repeatedly washed in phosphate-buffered saline, and finally resuspended at 5 x 108/ml in 0.01 M Tris, pH 8.5. The cells were then disrupted with a Biosonik III Sonifier, in 2-ml volumes for 45 s at 0 C, with frequent halts to avoid heating. This crude homogenate was high in nonspecific endonuclease activity. Centrifuging it in an SW 50.1 rotor at 40,000 rpm for 115 min at 5 C sedimented most of the nonspecific activity while leaving much of the activity for UV-irradiated DNA in the top half of the centrifuged solution. The





nonspecific activity seemed to be associated with faster sedimenting particles. For the experiments reported here, the top one-fourth of the centrifuged solution was recovered and frozen at -52 C in small samples for later assay. Figure la-d shows the sedimentation profiles of a representative set of neutral sucrose gradients of: (a) untreated RF I preparation; (b) RF I preparation exposed to 4,000 ergs of 254 nm UV per mm2; (c) unexposed RF preparation treated with extract at full concentration; and (d) UV-exposed RF preparation treated with the same extract. All samples were incubated for 25 min at 23 C, including samples (a) and (b) which contained buffer instead of extract. Some remaining nonspecific activity was indicated by the conversion of some unirradiated RF I to RF II (lc versus a). However, there was a much greater conversion of the UV-irradiated RF I to I

RFI \ F b /

(b) _


Endonuclease from Dictyostelium discoideum that attacks ultraviolet-irradiated deoxyribonucleic acid.

An endonuclease activity yielding single-strand breaks in ultraviolet light-irradiated phiX-174 RF I DNA in vitro has been detected in homogenates of ...
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