Absence of significant age-dependent increase of single-stranded DNA extracted from mouse liver nuclei

13, pp. 287-292. ~pPergamonVol. Press Ltd., 1978. Printed in Great Britain.

0531-5565/78/1001-0287502,00/0

. Geront.

ABSENCE OF SIGNIFICANT AGE-DEPENDENT INCREASE OF SINGLE-STRANDED D N A EXTRACTED FROM MOUSE LIVER NUCLEI ROGER G. DEAN and RICHARD G. CUTLER* Laboratory of Cellular and Molecular Biology, Gerontology Research Center, National Institute on Aging, National Institute of Health, PHS, U.S. Department of Health, Education and Welfare, 8¢thesda and the Baltimore City Hospitals, Baltimore, Maryland, U.S.A. (Received 7 February 1978)

INTRODUCTION MUCH age-dependent physiology and pathology could be explained by a gradual loss in cellular informational storage and transfer processes (Cutler, 1976a, 1976b, 1978). Such changes could result from an accumulation of physico-chemical alterations of chromatin. Our laboratory has undertaken a systematic study of the possible physico-chemical alterations that occur in chromatin and the corresponding protective and stabilizing processes that might underlie the different aging rates of mammalian species (Cutler, 1978). Recent results of another laboratory have indicated the presence and substantial agedependent increase in the fraction of single-stranded D N A contained in liver (Chetsanga et al., 1975), heart (Chetsanga et al., 1976), and brain (Chetsanga et al., 1977). The greatest increase was found in liver, where up to 25 % of the D N A in old mice was determined to be single-stranded. Such an alteration of D N A could have far-reaching implications for proper genetic function of a cell. These results were obtained using the single-stranded specific nuclease S~ isolated from Aspergillus oryzae. This nuclease has found widespread use as a structural probe for alterations in D N A double-stranded structure (Chan and Wells, 1974; Shenk et al., 1975) and in the removal of unhybridized regions in D N A ' R N A hybridization studies (Sutton, 1971). These applications are based on the extremely high specificity of this enzyme for singlestranded, as compared to double-stranded nucleic acids (Vogt, 1973; Ghangas and Wu, 1975; Hoffman and Collins, 1976). We sought to confirm the age-dependent accumulation of single-stranded D N A in mouse liver using the S~ nuclease digestion technique, isopycnic density gradient centrigufation, and thermal stability. No significant amount or age-dependent increase in single-stranded D N A was found. METHODS AND PROCEDURE Animals and DNA preparation

Male C57B1/6 mice of three age groups were used: young adult, 4.5-7.5; mature adult, 20; and old adult, 28-31 months. Mice were fed (Purina Chow) and watered adlibitum and were housed at constant temperature (24°C) with 12-h day-night cyclic lighting. The maximum life span potential of these mice is about 1200 days with a 50% mean survival of 750 days (Kunstyr and Leuenberger, 1975; Prashad and Cutler, 1976). Liver DNA was prepared as described by Chetsanga et al. (1976, 1977), as an improvement (Chetsanga, personal communication) over the method originally used with liver (Chetsanga et al., 1975). Each DNA preparation was from a single fresh mouse liver. DNA from young and old age groups were prepared together to minimize possible variation that might exist during a given extraction procedure. Mice were killed by cervical dislocation. The liver was removed, minced in ice-cold borate buffer (0.18 M NaCl, 0"02 M disodium *To whom reprint requests should be addressed. 287

288

R O G E R G . D E A N A N D R I C E I A R D Ci. C U T L E R

EDTA, 0-12 M boric acid and 0.042 M NaOH, pH 8'5), washed and homogenizcd in 10 vol. (w/v) borate buffer by 10 passes at 1500 rpm using a teflon-glass homogenizer having a clearance of 0.15 mm. The homogenate was filtered through 4 layers of gauze and centrifuged at 1000 :
Thermal melthlg profiles D N A was dialyzed overnight against 2.5 -~ 10 ' M NaCI and adjusted to a concentration of about 33 lagm/ml. Optical density at 260 nm and temperature were recorded continuously as a function of time using a Gilford 2000 spectrophotometer fitted with a thermal jacket, automatic blank compensator, and temperature probe.

Isopycnic density gradient centrifugation Cesium chloride gradients were prepared by adding 7.2 g of solid CsC1 to 5.66 ml of 0.01 M Tris, 0.01 M NaCI buffer solution containing 50 lag DNA, pH 7.5. The resultant volume of 7.5 ml was adjusted, if necessary, to a refractive index of 1.4002 by the addition of buffer or solid CsCI. Samples were placed in polyallomer tubes and centrifuged in a Beckman 50 Ti rotor at 106,500 :< g for 72 h at 25°C. Tubes were pierced and 0'15-ml fractions were collected at 0.45 ml/min using a peristaltic pump. Refractivc index and optical density at 260 nm were determined for selected samples.

S~ nuclease digestio, Digestion of DNA with S~ nuclease (Calbiochem) was in duplicate for both native and denatured mouse liver D N A samples. Denatured D N A was obtained by heating native D N A in boiling water for 10 min. then quickly cooling in ice. The reaction mixture for S~ nuclease digestion contained about 50 lag/ml D N A (native or denatured) in 1 ml of the Tris-NaCl buffer with 0"5 ml of 3 × KZS (0.3 M KCI, 0.3 mM ZnSO4, 0.075 M sodium acetate, pH 4.5) and 15 lag S~ nuclease added. Samples were incubated for 30 minutes at 37°C. After cooling on ice, 25 lag carrier DNA (calf thymus, Worthington) in 0.1 ml was added to each sample, followed by 0.5 ml of 2 N HCIO4. Samples were maintained on ice for 20 rain and then centrifuged at 9400 ~ g for 20 min. The fraction of acid-soluble material was determined by absorbance at 260 nm. Control samples were treated in the same manner, except that they were precipitated immediately with HCIO4 after the addition of S~ nuclease. RESULTS

Sl nuclease digestio, T a b l e I s u m m a r i z e s t h e r e s u l t s o f t h e S~ n u c l e a s e d i g e s t i o n o f e x p e r i m e n t s u s i n g t h e p u r i f i e d l i v e r D N A f r o m t h e t h r e e a g e g r o u p s ( y o u n g , m a t u r e a n d o l d ) o f mice. F o u r independent experiments were completed for each age group. The average percent of native D N A s u s c e p t i b l e t o $1 d i g e s t i o n o f t h e t h r e e a g e g r o u p s w a s f o u n d t o b e : y o u n g , 3 - 7 . 0 , m a t u r e , 4.7 ~ , a n d old, 4.1 ~ . T h e s t a n d a r d d e v i a t i o n s f o r t h e d a t a a r e less t h a n 1 ~ b u t give c o n s i d e r a b l e o v e r l a p b e t w e e n t h e t h r e e ages, t h u s s h o w i n g n o d i s t i n g u i s h i n g d i f f e r e n c e s in $1 s u s c e p t i b i l i t y w i t h age. T h e h e a t d e n a t u r e d c o n t r o l s a m p l e s s h o w n in T a b l e 1 d e m o n s t r a t e t h a t t h e $I n u c l e a s e a s s a y is w o r k i n g s a t i s f a c t o r i l y a n d c o n v e r t s t h e s e c o n t r o l s a m p l e s a l m o s t c o m p l e t e l y t o a c i d - s o l u b l e p r o d u c t s , as e x p e c t e d .

ABSENCE OF AGE-DEPENDENT INCBEASE OF SINGLE-STRANDED DNA FROM MOUSE LIVER NUCLEI

289

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from young (7 month) mice, (©) DNA from old (30 month) mice. Final hyperchromicity for old mice is 34"9% with Tm=63.6°C. Final hyperchromicity for young is 35.4% with Tm of 64.50C.

Thermal melting profiles Single strandedness in DNA preparations may also be detected by measuring the percent hyperchromicity obtained on thermal denaturation. Figure 1 shows the thermal melting profiles of liver DNA from 7.5 and 31-month old mice. Only slight differences are observed. Percent hyperchromicity for young and old mice differ by less than 1%, and the shapes of the curves are almost identical. The Tm's of young and old DNA show no significant difference but are about 6° lower than expected when compared to theoretical values based on GC content (Mandel and Marmur, 1968). These values do agree, however, with other Tm's cited for mouse liver DNA at similar ionic strengths (Herrman et al., 1975). CsC1 density gradient centr,fugation As a further characterization of liver DNA from young and old mice, buoyant densities were determined in CsCI gradients (Fig. 2). Presence of a significant fraction of singlestranded D N A would be expected to produce a banding pattern reflecting the higher density of the single-stranded regions,. The buoyant density for both young and old mouse liver DNA was found to be about 1.702 g/cm3, agreeing with published values for double-stranded mouse main-band DNA (Chun and Littlefield, 1963). The resolution level of our analysis was not sufficient to detect the well-known mouse satellite D N A peak at 1.691 g/cm3; but skewing toward the lower density portion of the gradient is evident in both the young and old mouse D N A samples. Overall, the differences in banding patterns between the young and old DNA's are minor and can be considered negligible. DISCUSSION We have examined the fraction of single-stranded DNA existing as a function of age in

290

ROGER G. DEAN AND RICHARD G. CUTLER TABLE I . S~ NUCLEASE DIGESTION OF D N A OBTAINED FROM MOUSE LIVER 260 nm absorbance of acid-soluble Denatured DNAt Percent of DNA sensitive to S,

Age (months)

material* Native DNA Denatured DNA

(corrected)

nuclease Native DNA Denatured D N A

4.5 5"0 6'5 7-5

0"025 0"028 0-020 0.020

0.671 0.700 0"653 0.672

0-705 0.668 0'693 0.669

20 20 20 20

0-034 0"026 0-034 0"032

0-664 0.642 0.659 0-681

28 28 30 31

0"025 0"029 0-025 0'035

0"691 0'690 0"674 0-681

0.673 0.675 0.667 0-669 Average 0"707 0-665 0.700 0'671

3'5 4.2 2-9 4.3 (3.7:!_0.66) 5. I 3.9 5-8 4.8 (4-7 ::~:0"57) 3'5 4.4 3"6 5' 1 (4.1 _~30.75)

Average

95.2 140.8

94-2 100-4 t98'6 ! 4"9) 98.6 95- I 98.8 98.6 (97"7:~: 1'8) 97"8 103.7 96'3 101 "4 (99-83~3.4)

Average *Corrected by subtracting absorption of zero-incubation time controls. tCorrected absorbance is obtained by assuming a 40 ~ hyperchromicity factor.

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FIG. 2. CsCI density gradient centrifugation of senescent (31 month) (O) and young (7-5 month) ( 0 ) mouse liver D N A .

mouse liver D N A by three types of measurements. The S, nuclease assay employed in these experiments is a direct and sensitive assay for single-stranded regions. Our investigations show no evidence for an age related increase. Instead, we only found a low and constant baseline level of about 4 o/, independent of age. The S, nuclease assay is capable of detecting as little as a 4 ~ difference in single-strandedness between age groups (approximately 2 times S.D.). We cannot rule out small age related changes in single-strandedness within this 4 variation. However, it is clear that large differences in single-strandedness could be detected - - b u t were not observed. This result is in agreement with Scheid e t al. (1975), who found no difference in single-stranded regions of D N A from pre- and postmenopausal human ovaries, but in sharp contrast with the results of Chetsanga et al. (1975), who showed that as much as 25 °/,, of the liver D N A in senescent mice was single-stranded.

ABSENCE OF AGE-DEPENDENT INCREASE OF SINGLE-STRANDED DNA FROM MOUSE LIVER NUCLEI

291

These negative findings with the SI nuclease assay are supported by the thermal melting profiles. Melting profiles of native DNA with single-stranded DNA present would be evident by a reduced hyperchromicity and/or a change in the thermal denaturation profile (Doty et al., 1959; Hirschman and Felsenfeld, 1966; von Hahn and Fritz, 1966). We have estimated that native DNA containing 10 ~ single-stranded regions would give a reduction in hyperchromicity of over 4 ~o which this assay could detect. Nevertheless, we found no significant difference in total hyperchromicity or profile. Gaubatz and Cutler (1978), using alkaline denaturation of mouse brain, spleen, and kidney DNA preparation, have found reduced DNA hyperchromicity in 30-month old mice. On the other hand, Russell et al. (1970) show no difference in thermal hyperchromicity of liver DNA from 2, 10 and 20-month old mice. These findings appear to be dependent on the method used to isolate the DNA and the possible differences that might exist in its purity. CsCl density gradient centrifugation also showed no evidence of single-stranded regions in the liver DNA of senescent mice. The banding patterns of senescent and young mouse DNA were essentially the same, which has previously been shown with cow thymus DNA (Pyhtila and Sherman, 1967) and mouse liver (Gaubatz and Cutler, 1978). The presence of extensive regions of single-stranded DNA (20~) in otherwise native DNA should cause a slight shift of the main band, peak to higher density and for a detectable shoulder neighboring the main band at higher density (Schildkraut et al., 1961). Chetsanga et al. (1976) have shown extensive broadening and skewing to the lighter region of CsCl gradients for senesceant mouse heart DNA, compared to young DNA. The broadening was thought to be due to a lower molecular weight for the senescent mouse DNA. No evidence of single-stranded DNA was found in the higher density region of the gradient in spite of the fact that their $1 nuclease assay indicated that 15 ~ of the senescent mouse heart DNA was single-stranded. In three independent assays of mouse liver DNA, we have observed no increased singlestrandedness with age. This is strong evidence in disagreement with the findings of Chetsanga. Although it is possible that his method of preparing DNA may yield variable amounts of single-strandedness, it is doubtful that this could be the source of our disagreement. Chetsanga basically used two different procedures in preparing DNA from three different mouse tissues. In all cases he found greater sensitivity of old mouse DNA to digestion by S~ nuclease. It would seem that the demonstration of single-strandedness does not depend heavily upon the procedure or tissue used. Although we did not use exactly the same procedure in preparing DNA from liver (Chetsanga et al., 1975), we did follow the procedures used for heart and brain (Chetsanga et al., 1976; Chetsanga et al., 1977). Therefore, we feel that our failure to show increased single-strandedness with age is not caused by selective loss of singlestranded regions or procedural artifact. However, as a further test against the possible selective loss of single-stranded regions, liver DNA was prepared using an entirely different procedure (Prashad and Cutler, 1976). This procedure gave higher yields of DNA (1.9 mg/g of tissue), but again we found no difference in $1 nuclease sensitivity for young and old mouse DNA. SUMMARY Possible age-dependent accumulation of physico-chemical changes in purified mouse liver DNA was investigated. Previous reports of another laboratory (Chetsanga et al., 1977) have indicated the presence and substantial age-dependent increase in the fraction of single-

292

R,)C,,

DEAN AND RICHARD G. CU'FLER

stranded regions in the D N A f r o m three mouse tissues. Using the S~ nuclease t ech n i q u e for detecting single-stranded D N A sequences, as well as isopycnic density gradient centrifugation and t h e r m a l stability analysis, we c a n n o t confirm this result and find no significant change in the d o u b l e - s t r a n d e d integrity o f purified mouse liver D N A . REFERENCES H. W. and WELLS, R. D, (1974) Nature, Lond. 252, 205. CHETSANGA,C. J., BOVD,V., PETERSON,L. and RUSHLOW,K. (1975) J. biol, Chem. 253, 130. CHETSANGA,C. J., TOTTLE, M. and JACOBONI,A. (1976) Life Sci. 18, 1405. Cr~ETSANGA,C. J., TUTTLE, M., JACOBONI,A. and JOttNSON, C. (1977) Riochim. biophys, Acta 474, 180. CHUN, E. H. L. and LITTLEFIELD,J. W. (1963) J. molec. Biol. 7, 246. CUTLER, R. G. (1976a) In: htterdiscipl. Topics Gerontology (Edited by R. G. CUTLER),p. 83. Karger, Basel. CUTLER, R. G. (1976b) In: Protein and Other Adducts to DNA : Their Significance to Aging. Carcinogenesis, and Radiation Biology (Edited by K. C. SMITH), p. 443. Plenum Press, New York. CUTLER,R. G. (1978) In: Genetic Effects on Aghtg (Edited by D. BERGSMA,D. E. HARRmONand N. W. PAUL), pp. 463~,98. Alan K. Liss, New York. In press. DOTY, P., BOEDTKER,H.. FRESCO,J. R., HASELKORN,R. and Lrlq, M. (1959) Proe. hath. Acad. Sci., U.S.A. 45, 482. GAUBATZ,G. S. and CUTLER, R. G. (1978) Gerontology 24, 179. GHANGAS, G. S. and Wu, R. (1975) J. biol. Chem. 250, 4601. HERRMAN, R. L., DOWLIN(;, L., RUSSELL,A. P. and BICK, M. D. (1975) Mech. Age. Devl. 4, 181. HIRSCHMAN,S. Z. and FELSENFELD,G. (1966) J. ttlolec. Biol. 16, 347, HOFFMAN, L. M. and COLLINS,J. M. (1976) Nature, Lond. 260, 642. KUNSrYR, 1. and LEtJENaERGER,H. W. (1975) J. Geront. 30, 157. MANDEL, M. and MARMUR,J. (1968) ,~lethods Enzym. (B) 12, 195. PRASHAD, N. and CUTLER, R. G. (1976) Biochim. biophys. Acta 418, I. PHYTILA, M. J. and SHERMAN,F. G. (1967) Fedu. Proc. 26, 667. RUSSELL,A. P., DOWLING, L. E. and HERRMAN,R. L. (1970) Gerontologia 16, 159. SCHEID, B., PEDRINAN,L., LU, T. and NEt.SON,J. H. (1975) Biochem. biophys. Re~'. Commun. 66, 1131. SCHILDKRAUT,C. L., MARMUR,J., DOTY, P. (1961) J. molec. Biol. 3, 595. SHENK, T. E., RHODES,C., RIGBY, P. W. J. and BERG, P. t 1975) Proc. natn. Aead. Sci., U.S.A. 72, 989. SUTTON, W. D. (1971) Bioehim. biophys. Acta 240, 522. VOGT, V. M. (1973) Eur. J. Biochem. 33, 192. VON HAHN, H. P. and FRrrz, E. (1966) Gerontologia 12, 237. CHAN,