Mutation Research, D NA Repair, 255 (1991) 31-38 © 1991 Elsevier Science Publishers B.V. 0921-8777/91/$03.50 ADONIS 092187779100078H
31
MUTDNA 06441
Alteration of a nuclease in Fanconi anemia Kengo Sakaguchi
a,.,
P a u l V. H a r r i s a, C a r o l R y a n b, M a n u e l B u c h w a l d b,c a n d J a m e s B. B o y d a
Department of Genetics a, University of California, Davis, CA 95616 (U.S.A.) and Research Institute b, Hospital for Sick Children, Department of Medical Genetics ~, Medical Biophysics c, University of Toronto, 555 University Avenue, Toronto, Ont. M5G1X8 (Canada) (Received 3 September 1990) (Revision received 2 January 1991) (Accepted 25 January 1991)
Keywords: Fanconi anaemia; Complementation group A; Nuclease; Isoelectric focusing
Summary Fanconi anemia is a cancer-prone disease characterized by progressive loss of blood cells, skeletal defects and stunted growth. Studies of a nuclease acting on double-stranded DNA have revealed an enzyme alteration in cells derived from Fanconi patients. A particulate fraction isolated from cultured human lymphoblasts and fibroblasts was solubilized with detergent and subjected to isoelectric focusing. Nuclease activity observed in four normal cell lines bands in a pH gradient with a p l of 6.3. Four cell lines belonging to complementation group A exhibit an increase in the pI of that nuclease to 6.8. These observations provide a new diagnostic for this disorder. Analysis of this enzyme in tetraploid cultures derived from fusion of normal and Fanconi cells suggest that the normal phenotype is dominant. That observation supports the hypothesis that the Fanconi A gene is required for modification of the nuclease pI. Definition of the molecular basis of this enzyme alteration should provide insight into the primary genetic lesion in this disorder.
Fanconi anemia (FA) is a rare autosomal recessive disease associated with a complex spectrum of clinical symptoms (see Schroeder-Kurth et al., 1989). Patients suffering from this disorder are
* Present address: Department of Applied Biological Science, Faculty of Science and Technology, Science University of Tokyo, Noda-shi, Chiba-ken, Tokyo (Japan). Correspondence: Prof. James B. Boyd, Department of Genetics, University of California, Davis, CA 95616 (U.S.A.).
Abbreviations: FA, Fanconi anemia; FA-A, complementation group A of Fanconi anemia.
predisposed to leukemia and generally die from bone-marrow failure by late childhood. The extensive variability observed in this disorder has been partially resolved by the use of cell fusion to identify at least two different complementation groups (Duckworth-Rysiecki et al., 1985; Zakrzewski and Sperling, 1980). Cells from the A complementation group exhibit a reduced capacity to recover normal levels of semiconservative DNA synthesis after mutagen treatment (Moustacchi et al., 1987). Further evidence for the existence of a fundamental cellular defect in this disorder defives from studies designed to suppress phenotypic defects at the cellular level. In those studies FA
32 phenotypes have been reversed both by microinjection of cell extracts (GiSk and Wunder, 1987) or RNA (Digweed and Sperling, 1989) from normal cells and by transfection with normal human D N A (Diatloff-Zito et al., 1986; Shaham et al., 1987). Extensive cytological studies of cell cultures derived from these individuals has provided further evidence for a defect in DNA metabolism. Together with Bloom's syndrome and ataxia telangiectasia, FA is associated with a high incidence of spontaneous chromosomal aberrations. Peripheral blood lymphocytes and established cell cultures both exhibit elevated chromatid breaks and gaps, although chromosome breaks are rare (Schroeder, 1982). That phenomenon is strongly potentiated by exposure of the cells to caffeine (Pincheira et al., 1988) and D N A cross-linking agents such as mitomycin C, diepoxybutane, nitrogen mustard and cisplatin (see Auerbach et al., 1989). The growth capacity of FA lymphoblasts is strongly reduced by those mutagens (Ishida and Buchwald, 1982) as well as by psoralen monoadducts (Averbeck et al., 1988). The induction of sister-chromatid exchange that is normally induced by bifunctional agents is also strongly suppressed in FA cells (Latt et al., 1975). Recent studies of mutagen sensitive Drosophila strains have revealed a striking parallel between FA and the mus308 mutants of that organism (Boyd et al., 1990). In both the Drosophila and human disorders the cellular phenotype is characterized by selective hypersensitivity to D N A cross-linking agents and by elevated frequencies of spontaneous chromosomal aberrations. In addition, mutant mus308 °2 cells of Drosophila fail to recover semiconservative D N A synthesis following mutagen treatment (Brown and Boyd, 1981) as do ceils from the A complementation group of FA (Moustacchi et al., 1987). Biochemical and genetic studies of the mus308 mutants have revealed an enzyme alteration that is revealed as an increased p I (Boyd et al., 1990; Sakaguchi et al., 1990). This report documents an analogous defect in cells assigned to the A complementation group of FA. Materials and methods
Cell lines and cell culture. Lymphoblast fines from normal individuals and patients with FA
were derived as described (Ishida and Buchwald, 1982). SV40-transformed fibroblasts from a normal individual (GM00637D) and from a patient with FA (GM06914A) were purchased from the Human Genetic Mutant Cell Repository (Camden, N J). The FA cell line GM06914A has been assigned to the A complementation group (Duckworth-Rysiecki et al., 1986). The control lymphoblast line K562 (GM512) was obtained from the Human Genetic Mutant Cell Repository and the fibroblast line AVneo from E.C. Friedberg. Lymphoblasts were cultured either in RPMI Media 1640 (Gibco) supplemented with 12% fetal bovine serum (Gibco) or in MEM supplemented with non-essential amino acids and 12% fetal bovine serum. Fibroblasts were maintained in alphaMEM with Hanks' salts (Gibco) supplemented with 15% fetal bovine serum. All cultures were maintained at 37°C in a humidified atmosphere containing 5% CO z. Culture medium was changed twice a week. Analysis of mutagen sensitivity. Sensitivity of selected lymphoblast cell lines to D N A crosslinking agents was monitored with a modification of the procedure described by Ishida and Buchwald (1982). Single-cell suspensions derived from exponentially growing cultures were distributed to 35 mm culture dishes at a density of 0.5 x 10 6 cells/ml. One day later the cells were treated with mutagen dissolved in physiological saline. 4 - 5 days after mutagen treatment metabolically active cells identified by dye exclusion were counted. Nuclease assay. The standard assay reaction of 200 /xl contained 150 /d of 50 m M acetate at pH 5.5, 5 mM MgC12, 1 mM DTT, 15% glycerol, 0.1% Triton X-100, 5 mM ATP and 5 /~g [3H]E. coli DNA (about 12 500 cpm/txg DNA). The reaction was initiated by addition of 50/xl of enzyme extract and terminated 3-5 h later by cooling and the addition of 200 t~l of 5 m g / m l bovine serum albumin and 0.6 ml of 10% ( w / v ) trichloroacetic acid. Each fraction was centrifuged and the radioactivity in the supernatant was determined by scintillation counting. The assay is linear with time between 0% and at least 50% solubilization of the substrate.
33
Organelle isolation. A particulate fraction was prepared from 1-3 x 107 cells hypotonically swollen for 10 rain in 5 ml of TE (10 mM Tris-HC1, pH 7.5, 1 mM EDTA). Following addition of 1 ml of 1.45 M sucrose in TE, the cells were homogenized with 10 strokes in a Dounce homogenizer (Kontes, B pestle). The homogenate was centrifuged at 1700 × g for 5 min and the resulting supernate was further spun at 2000 x g for 5 min. The pellet derived by centrifuging that supernate at 12 000 x g for 20 min was suspended in 1.5 ml lysis buffer (50 mM Tris-HC1, pH 7.4, 1 mM EDTA, 5 mM fl-mercaptoethanol, 15% glycerol, 0.8 M NaC1, 0.4% Triton X-100, 1 mM PMSF and 1/~g/ml each of pepstatin and leupeptin). Following sonication for 5 sec, the sample was again centrifuged at 1 2 0 0 0 × g for 20 min, and the supernatant was analyzed by isoelectric focusing. In a restricted number of cases organelles were recovered from a sucrose step gradient. Isoelectric focusing. The isoelectric focusing procedure has been described (Sakaguchi and Boyd, 1985). Focusing was performed in ll0-ml gradients of 5-40% sucrose containing 0.75% (v/v) carrier ampholyte (pH ranges; 3.5-10 or 5-8), 5% glycerol, 5 mM fl-mercaptoethanol, 0.4% Triton X-100 and 1/~g/ml each of leupeptin and pepstatin. The gradients were formed at 350 V for 40 h at 4°C and collected in 2-ml fractions. In selected experiments cells from mutant and control lines were mixed prior to analysis. Equivalent results were obtained when mixing was performed at each of these stages.
ously documented (Ishida and Buchwald, 1982), the identity of selected cell cultures was verified by determining sensitivity to nitrogen mustard and mitomycin C (Table 1). That analysis reveals a 2-fold increase in the sensitivity of HSC99 cells to both mutagens. Similarly the HSC72 cell line is 4-fold more sensitive to these agents. These sensitivities are approximately equivalent to those observed by Matsumoto et al. (1989) and thus serve to establish that a Fanconi anemia phenotype is being expressed under the experimental conditions employed in this study. The fact that these values are lower than those measured earlier by Ishida and Buchwald (1982) is probably attributable to the different criteria employed to assay inhibition of cell growth.
Identification of an organelle DNAase. Guided by previous studies of the model organism Drosophila (Boyd et al., 1990), biochemical analysis of these Fanconi lines has focused on a deoxyribonuclease derived from a particulate fraction composed predominantly of mitochondria and lysosomes. Preliminary characterization of that activity has established a pH optimum of 5.5 and a 4-fold preference for native over denatured DNA. The activity is insensitive to N-ethylmaleimide but is increased in the presence of magnesium and TABLE 1 SENSITIVITY OF LYMPHOBLAST CELL LINES TO NITROGEN MUSTARD (HN2) AND MITOCYCIN C (MMC) Mutagen
Results
Cell cultures. This study focuses principally on the three lymphoblast cell lines HSC72, HSC99 and HSC527, which were derived from unrelated individuals who have been identified with the A complementation group of FA (DuckworthRysiecki et al., 1985, 1986). Confirmatory results have also been obtained with the fibroblast line GM6914 that was derived from an effected sibling of the individual who gave rise to HSC527 (Duckworth-Rysiecki et al., 1986). Although hypersensitivity of the mutant cell lines to DNA cross-linking agents has been previ-
Cell line
Mutagen concentration Control
Fanconi group A
HSC93
HSC99
HSC72
HN2 (nM) Relative sensitivity
490+ 119 (6)
197+89 (5)
113+23 (3)
MMC(nM) Relative sensitivity
105+ 33(5)
1.0
1.0
2.5 54+15(4) 1.9
4.3 24+ 3(2) 4.4
The indicated mutagen concentrations reduce cell viability by 50% relative to untreated cultures. Those concentrations are presented with standard deviations and the number of determinations in brackets. Relative mutagen sensitivity is the control value divided by the corresponding control or mutant value.
34 A T P ( u n p u b l i s h e d observations). The p r i n c i p l e exp e r i m e n t a l p a r a m e t e r a s s a y e d in this s t u d y is the p I o f that enzyme. T h a t value is m o n i t o r e d b y i d e n t i f y i n g the e n z y m e p o s i t i o n in a p H g r a d i e n t following p r o t e i n f r a c t i o n a t i o n b y isoelectric focusing. T h e t o p p a n e l of Fig. 1 reveals the d i s t r i b u t i o n of nuclease activity in a p H g r a d i e n t e x t e n d i n g f r o m 5 to 8. T h e p e a k of activity rec o v e r e d f r o m n o r m a l cells occurs at a p I of a b o u t 6.3.
5
6
7 _--
~"
.P'4
8
::.~
GM637D
...............
GM6914
I
•
:: . . ::: ...
..............I ).............)
Aberrant p I in Fanconi anemia cells.
The center p a n e l of Fig. 1 reveals that the e n z y m e f r o m a F A - A f i b r o b l a s t cell line exhibits a p I that is a b o u t 0.5 units higher t h a n that of the control. T h e s h a d e d b a r on the left indicates that the range of the p e a k p I values o b s e r v e d for all a n a l y z e d c o n t r o l cell lines varies b e t w e e n 6.2 a n d 6.4. T h e s h a d e d b a r on the fight reveals that the p I values o b s e r v e d in the four a v a i l a b l e F A - A lines a n d their derivatives v a r y b e t w e e n 6.7 a n d 7.0. Since these two ranges d o n o t overlap, this p a r a m e t e r p r o v i d e s a reliable d i a g n o s t i c of the F A - A defect. A s shown in the b o t t o m p a n e l o f that figure, the u n i q u e i d e n t i t y of the c o n t r o l a n d F A e n z y m e s is e s t a b l i s h e d b y their s e p a r a t i o n o n a single focusing gradient. I n that e x p e r i m e n t cells f r o m the two cultures were m i x e d p r i o r to cell d i s r u p t i o n a n d e n z y m e fractionation. T h a t c o n t r o l excludes fract i o n a t i o n errors a n d reduces the l i k e l i h o o d that the o b s e r v e d p I difference is d u e to artifacts introd u c e d b y p r o t e o l y s i s or aggregation. T h e m i x i n g c o n t r o l also establishes that a n y m o d i f i c a t i o n factors, which might b e r e s p o n s i b l e for c o n v e r t i n g one e n z y m e f o r m to another, are n o t active u n d e r the c o n d i t i o n s e m p l o y e d for e n z y m e e x t r a c t i o n a n d f r a c t i o n a t i o n . If that were so, one e n z y m e f o r m w o u l d be c o n v e r t e d to the o t h e r d u r i n g f r a c t i o n a t i o n following cell mixing. D a t a in Fig. 2 e x t e n d the initial o b s e r v a t i o n s m a d e in f i b r o b l a s t s to a series of l y m p h o b l a s t cell lines. In that figure d a t a o b t a i n e d with two different c o n t r o l cell lines are p r e s e n t e d in the t o p p a n e l s for direct c o m p a r i s o n with d a t a f r o m the three available F A - A cell lines. I n each case the p I of the F A nuclease is r e p r o d u c i b l y higher t h a n that of the control. A n a l y s i s of m i x t u r e s of c o n t r o l a n d F A - A cells a g a i n c o n f i r m the u n i q u e i d e n t i t y of these two e n z y m e forms. A consistent shift in
I !:::::::::,::!
Mix
5
!::::::::::::!
:.:::::::::::" : : : " :
6 7 pH Within Gradient
8
Fig. 1. An organeller nuclease from an SV40 transformed FA fibroblast cell line exhibits an altered pl. A particulate fraction was prepared from control (GM00637D) and FA-A (GMO6914A) fibroblast cell lines. Protein was solubilized from the isolated organelles by sonication as described in Materials and Methods. Following centrifugation, the extracts were subjected to preparative isoelectric focusing and fractions were assayed for nuclease activity. In the bottom panel, an equal number of cells from those two lines were mixed prior to extraction. Nuclease activity in the peak fractions varied from 875 to 927 cpm solubilized per h. The shaded bar on the left designates the range of pl observed in the four different control cell lines assayed in this study (10 determinations). The shaded bar on the right represents the range of pI values found in the four FA-A lines assayed in this study (6 determinations).
p I has therefore been d o c u m e n t e d in two cell t y p e s d e r i v e d f r o m three d i f f e r e n t families affected with m u t a t i o n s in the F A - A gene.
35
The normal p I is expressed as a dominant trait. In both Fanconi anemia in man (DuckworthRysiecki et al., 1986) and in the mus308 mutants of Drosophila (Boyd et al., 1990) cellular hypersensitivity to D N A cross-linking agents is a recessive trait. In contrast, the wild-type form of the nuclease in Drosophila is dominant (Sakaguchi et al., 1990). That result indicates that the Drosophila gene is indirectly responsible for alteration of the nuclease p l rather than directly encoding a nuclease polypeptide. The question of dominance has been investigated in the FA-A disorder with a tetraploid cell line derived by cell fusion. That line was previously generated in complementation studies of FA (Duckworth-Rysiecki et al., 1985, 1986). D a t a presented in Fig. 3 reveal that a cell line generated by fusing the FA-A cell line HSC72 with the control lymphoblast line HSC93 expresses the control pattern of activity. This result suggests that the normal p l is dominant and 5
6
7
85
6
7
85
6
6
7
85
6
7
85
6
7
iiiiiii I000 "
~
iiiiiiii 500" ;:::;;:::
61 4
iiiiiiiii ~i!ii'i , 5 6 7 pH within gradient
8
Fig. 3. Nuclease analysis following cell fusion, A tetraploid cell line generated by fusion of the FA-A cell line HSC72 and the control cell line HSC93 was analyzed by isoelectric focusing as described in Fig. I.
thereby adds support to the hypothesis that the FA gene is required for the nuclease to assume the lower p I form.
Discussion
7
"r.
5
1500
8
pH within gradient Fig. 2. Alteration of the nuclease pI in lymphoblast cell lines of FA-A cultures. Nuclease was fractionated by isoeleetric focusing and assayed as described in Fig. 1. Control patterns are presented along the top row for comparison. The control data generated with K562 ceUswas obtained with a particulate fraction recovered from a discontinuous sucrose gradient. The mixing experiments presented in the lower panels were performed by combining an equal number of cells from the two cultures depicted above in each case. As in Fig. 1, the shaded bar on the left designates the range of pl observed in four different control cell lines and that on the right represents the range of p l values determined for all FA-A lines. Nuclease activity in the peak fractions varied from 1455 to 3375 c p m solubilized per h.
This study provides a direct association between the modification of an organelle nuclease activity and the A complementation group of FA. The consistency of the control p I observed in several different cell types strengthens the generality of the control observation. An increase in p I has been observed in one fibroblast cell line and three lymphoblast lines derived from FA-A patients. The results of the mixing experiments support the supposition that the different enzyme forms have unique identities and are not due to artifacts g e n e r a t e d d u r i n g p r e p a r a t i o n or fractionation of the enzyme. Since two of the individuals donating these cells are siblings (Duckworth-Rysiecki et al., 1986), this phenotype has been documented for three independently investigated families. Cell fusion studies have also identified two cell lines that fall outside of the A complementation group of FA (DuckworthRysiecki et al., 1985). A parallel analysis of the nuclease in those lines has been initiated (Sakaguchi, unpublished), but an understanding of the results must await the completion of complementation tests that are currently being conducted. The hypothesis that the FA-A gene is required for modification of the nuclease p I is supported
36
by parallel studies of the mus308 mutants of Drosophila. Those mutants, whose study motivated this investigation, are analogous to FA-A in that they exhibit hypersensitivity to D N A cross linking agents without expressing a strong sensitivity to monofunctional alkylating agents (Boyd et al., 1990). Like FA-A cells, the Drosophila mutants also exhibit an elevated level of spontaneous chromosome aberrations and a failure to recover D N A synthesis following mutagen treatment (see Schroeder-Kurth et al., 1989; Gatti et al., 1984; Brown and Boyd, 1981). At least four independently derived mutations at that locus result in a shift in nuclease p I that is analogous to that reported here for FA-A (Boyd et al., 1990). Because independent structural gene mutations are unlikely to produce precisely the same alteration in protein charge, it is probable that these mutations in both organisms are responsible for altering the p I of a nuclease polypeptide. Our demonstration that the normal phenotype is dominant in fusions between FA-A and normal cells (Fig. 3, Table 1) is consistent with that hypothesis. If the FA-A gene were to directly encode a polypeptide of the nuclease, codominant expression rather than dominance would be expected in that situation. Attempts are therefore being made to identify in vitro conditions that will permit conversion of the mutant enzyme form to that of the control cells in an effort to identify the postulated modification factor. Preliminary studies suggest that the two activities may be differentially phosphorylated. A potentially homologous enzyme has also been analyzed in nine recently isolated Chinese hamster cell lines that are hypersensitive to DNA crosslinking agents (Zdzienicka et al., in press). Two of those lines have been shown to exhibit an enzyme modification analogous to that observed in the Drosophila and FA cells (unpublished; this report). Observations of an analogous nuclease defect in cross-link sensitive mutants in a third organism further strengthens the correlation between that phenotype and the aberrant nuclease p l reported here. That result, therefore, further increases the rationale for investigation of the altered nuclease p l in FA-A. This study focuses on a nuclease that is recovered from a particulate fraction with sedimentation characteristics of mitochondria and lyso-
somes. Electron microscopic observations (Singson, unpublished observations) have confirmed the prevalence of mitochondria in that fraction. An apparently analogous Drosophila enzyme has been shown by differential fractionation of organelles on continuous Ficoll gradients to be associated specifically with mitochondria (Sakaguchi et al., 1990). Further studies are needed, however, to establish whether mitochondria are the exclusive repository of this enzyme form in either Drosophila or man. Given that the enzyme is at least partially associated with subcellular organelles, it is appropriate to consider whether it can be identified with any previously studied organeller nucleases. Although a definitive comparison will depend upon current efforts to purify and characterize this enzyme, present evidence fails to establish a clear analogy with any previously isolated mammalian enzyme. It is probably not the classical lysosomal acid-active nuclease (Yamanaka et al., 1974), because it shares many properties with a Drosophila enzyme that is present in mutants lacking the putative lysosomal activity (Boyd et al., 1990). Properties of available crude preparations also do not correspond to those of two mitochondrial nucleases that have recently been characterized. A mitochondrial activity recovered from mouse cells by Tompkinson and Linn (1986) has no activity on native DNA, which is the substrate utilized in this study. Likewise a mitochondrial nuclease isolated from bovine, rat and man (Cummings et al., 1987; Low et al., 1988) is active at pH 7 - 8 whereas the enzyme under study here is not. It is, therefore, possible that the nuclease perturbed in FA cells represents a new enzyme form, although contaminants in current preparations could be obscuring its relationship to previously identified enzymes. Similarities between the phenotype of the mus308 mutants in Drosophila and FA in man prompted the current investigation which has further extended that analogy through observations of a nuclease modification in FA-A cells. Identification of a potentially common function in these two diverse multicellular eukaryotes opens the possibility for a genetic analysis of the Fanconi A function in Drosophila. In an effort to exploit that opportunity, efforts are currently underway to clone and characterize the Drosophila mus308
37 gene. T h a t a p p r o a c h will u l t i m a t e l y p e r m i t a deftnitive definition of the relationship between that g e n e a n d t h e F A - A gene.
Acknowledgements W e are g r a t e f u l to D r s . A. G a n e s a n , C. L a m b e r t a n d M . A . D i e c k m a n n for s u p p l y i n g cell cultures. D r s . I.R. L e h m a n a n d W. S i e d e p r o v i d e d h e l p f u l discussions. D r . E . C . F r i e d b e r g g e n e r o u s l y provided resources and encouragement during a s a b b a t i c a l l e a v e o f J.B.B. in his l a b o r a t o r y . F i n a n cial s u p p o r t w a s p r o v i d e d b y t h e U . S . D e p a r t m e n t o f E n e r g y (EV70210), t h e U n i t e d S t a t e s P u b l i c Health Service (GM32040), The Fanconi Anemia R e s e a r c h F u n d , Inc. a n d the M e d i c a l R e s e a r c h Council of Canada (MT7220).
References Auerbach, A.D., R. Ghosh, P.C. Pollio and M. Zhang (1989) Diepoxybutane test for prenatal and postnatal diagnosis of Fanconi anemia, in: Fanconi Anemia: Clinical, Cytogenetic and Experimental Aspects, Springer, Berlin, pp. 71-82. Averbeck, D., D. Papadopoulo and E. Moustacchi (1988) Repair of 4,5',8-Trimethylpsoralen plus light-induced DNA damage in normal and Fanconi's anemia cell lines. Cancer Res., 48, 2015-2020. Boyd, J.B., K. Sakaguchi and P.V. Harris (1990) The mus308 mutants of Drosophila exhibit hypersensitivity to DNA cross-linking agents and are defective in a deoxyribonuclease, Genetics, 125, 813-819. Brown, T.C., and J.B. Boyd (1981) Abnormal recovery of DNA replication in ultraviolet-irradiated cell cultures of Drosophila melanogaster which are defective in DNA repair, Mol. Gen. Genet., 183, 363-368. Cummings, O.W., T.C. King, J.A. Holden and R.L. Low (1987) Purification and characterization of the potent endonuclease in extracts of bovine heart mitochondria, J. Biol. Chem., 262, 2005-2015. Diatloff-Zito, C., D. Papadopoulo, D. Averbeck and E. Moustacchi (1986) Abnormal response to DNA crosslinking agents of Fanconi anemia fibroblasts can be corrected by transfection with normal human DNA, Proc. Nail. Acad. Sci. (U.S.A.), 83, 7034-7038. Digweed, M., and K. Sperling (1989) Identification of a HeLa mRNA fraction which can correct the DNA-repair defect in Fanconi anaemia fibroblasts, Mutation Res., 218, 171177. Duckworth-Rysiecki, G., C.A. Cornish, C.A. Clarke and M. Buchwald (1985) Identification of two complementation groups in Fanconi anemia, Somat. Cell Mol. Genet., 11, 35-41.
Duckworth-Rysiecki, G., L. Toji, J. Ng, C. Clarke and M. Buchwald (1986) Characterization of a Simian virus 40transformed Fanconi anemia fibroblast cell line, Mutation Res., 166, 207-214. Gatti, M., S. Pimpinelli, C. Bove, B.C. Baker, D.A. Smith, A.T.C. Carpenter and P. Ripoll (1984) Genetic control of mitotic cell division in Drosophila melanogaster, in: Genetics: New Frontiers, Oxford and IBS Publishing Co., New Delhi, pp. 193-204. GiSk, M.M., and E. Wunder (1987) Microinjection of normal cell extracts into Fanconi anemia fibroblasts corrects defective scheduled DNA synthesis recovery after 8-methoxypsoralen plus UVa treatment, Human Genet., 75, 350-355. Ishida, R., and M. Buchwald (1982) Susceptibility of Fanconi's anemia lymphoblasts to DNA-cross-linking and alkylating agents, Cancer Res., 42, 4000-4006. Latt, S.A., G. Stetten, L.A. Juergens, G.R. Buchanan and P.S. Gerald (1975) Induction by alkylating agents of sister chromatid exchanges and chromatid breaks in Fanconi's anemia, Proc. Natl. Acad. Sci. (U.S.A.), 72, 4066-4070. Low, R.L., J.M. Buzan and C.L. Couper (1988) The preference of the mitochondrial endonuclease for a conserved sequence block in mitochondrial DNA is highly conserved during mammalian evolution, Nucl. Acids Res., 16, 64276445. Matsumoto, A., J.-M.H. Vos and P.C. Hanawalt (1989) Repair analysis of mitomycin C-induced DNA crosslinking in ribosomal RNA genes in lymphoblastoid cells from Fanconi's anemia patients, Mutation Res., 217, 185-192. Moustacchi, E., D. Papadopoulo, C. Diatloff-Zito and M. Buchwald (1987) Two complementation groups of Fanconi's anemia differ in their phenotypic response to a DNA-crosslinking treatment, Human Genet., 75, 45-47. Pincheira, J., M. Bravo and J.F. Lopez-Saez (1988) Fanconi's anemia lymphocytes: effect of caffeine, adenosine and niacinamide during G 2 prophase, Mutation Res., 199, 159165. Sakagnchi, K., and J.B. Boyd (1985) Purification and characterization of a DNA polymerase fl from Drosophila, J. Biol. Chem., 260, 10406-10411. Sakaguchi, K., P.V. Harris, R. van Kuyk, A. Singson and J.B. Boyd (1990) A mitochondrial nuclease is modified in Drosophila mutants (mus308) that are hypersensitive to DNA crosslinking agents, Mol. Gen. Genet., 224, 333-340. Schroeder, T.M. (1982) Genetically determined chromosome instability syndromes, Cytogenet. Cell. Genet., 33, 119-132. Schroeder-Kurth, T.M., A.D. Auerbach and G. Obe (1989) Fanconi anemia: Clinical, Cytogenetic and Experimental Aspects, Springer, Berlin. Shaham, M., B. Adler, S. Ganguly and R.S.K. Chaganti (1987) Transfection of normal human and Chinese hamster DNA corrects diepoxybutane-induced chromosomal hypersensitivity of Fanconi anemia fibroblasts, Proc. Natl. Acad. Sci. (U.S.A.), 84, 5853-5857. Tompkinson, A.E., and S. Lima (1986) Purfication and properties of a single strand-specific endonuclease from mouse cell mitochondria, Nucleic Acid. Res., 14, 9579-9593.
38 Yamanaka, M., Y. Tsubota, M. Anai, K. Ishimatsu, M. Okumura, S. Katsuki and Y. Takagi (1974) Purification and properties of acid deoxyribonucleases of human gastric mucosa and cervix uteri, J. Biol. Chem., 249, 3884-3889. Zakrzewski, S., and K. Sperling (1980) Genetic heterogeneity
of Fanconi's anemia demonstrated by somatic cell hybrids, Hum. Genet., 56, 81-84. Zdzienicka, M.Z., J.W.I.M. Simons and P.H.M. Lohman (1990) DNA Repair Mechanisms and Their Biological Implications In Mammalian Cells, Plenum, New York, in press.
31
MUTDNA 06441
Alteration of a nuclease in Fanconi anemia Kengo Sakaguchi
a,.,
P a u l V. H a r r i s a, C a r o l R y a n b, M a n u e l B u c h w a l d b,c a n d J a m e s B. B o y d a
Department of Genetics a, University of California, Davis, CA 95616 (U.S.A.) and Research Institute b, Hospital for Sick Children, Department of Medical Genetics ~, Medical Biophysics c, University of Toronto, 555 University Avenue, Toronto, Ont. M5G1X8 (Canada) (Received 3 September 1990) (Revision received 2 January 1991) (Accepted 25 January 1991)
Keywords: Fanconi anaemia; Complementation group A; Nuclease; Isoelectric focusing
Summary Fanconi anemia is a cancer-prone disease characterized by progressive loss of blood cells, skeletal defects and stunted growth. Studies of a nuclease acting on double-stranded DNA have revealed an enzyme alteration in cells derived from Fanconi patients. A particulate fraction isolated from cultured human lymphoblasts and fibroblasts was solubilized with detergent and subjected to isoelectric focusing. Nuclease activity observed in four normal cell lines bands in a pH gradient with a p l of 6.3. Four cell lines belonging to complementation group A exhibit an increase in the pI of that nuclease to 6.8. These observations provide a new diagnostic for this disorder. Analysis of this enzyme in tetraploid cultures derived from fusion of normal and Fanconi cells suggest that the normal phenotype is dominant. That observation supports the hypothesis that the Fanconi A gene is required for modification of the nuclease pI. Definition of the molecular basis of this enzyme alteration should provide insight into the primary genetic lesion in this disorder.
Fanconi anemia (FA) is a rare autosomal recessive disease associated with a complex spectrum of clinical symptoms (see Schroeder-Kurth et al., 1989). Patients suffering from this disorder are
* Present address: Department of Applied Biological Science, Faculty of Science and Technology, Science University of Tokyo, Noda-shi, Chiba-ken, Tokyo (Japan). Correspondence: Prof. James B. Boyd, Department of Genetics, University of California, Davis, CA 95616 (U.S.A.).
Abbreviations: FA, Fanconi anemia; FA-A, complementation group A of Fanconi anemia.
predisposed to leukemia and generally die from bone-marrow failure by late childhood. The extensive variability observed in this disorder has been partially resolved by the use of cell fusion to identify at least two different complementation groups (Duckworth-Rysiecki et al., 1985; Zakrzewski and Sperling, 1980). Cells from the A complementation group exhibit a reduced capacity to recover normal levels of semiconservative DNA synthesis after mutagen treatment (Moustacchi et al., 1987). Further evidence for the existence of a fundamental cellular defect in this disorder defives from studies designed to suppress phenotypic defects at the cellular level. In those studies FA
32 phenotypes have been reversed both by microinjection of cell extracts (GiSk and Wunder, 1987) or RNA (Digweed and Sperling, 1989) from normal cells and by transfection with normal human D N A (Diatloff-Zito et al., 1986; Shaham et al., 1987). Extensive cytological studies of cell cultures derived from these individuals has provided further evidence for a defect in DNA metabolism. Together with Bloom's syndrome and ataxia telangiectasia, FA is associated with a high incidence of spontaneous chromosomal aberrations. Peripheral blood lymphocytes and established cell cultures both exhibit elevated chromatid breaks and gaps, although chromosome breaks are rare (Schroeder, 1982). That phenomenon is strongly potentiated by exposure of the cells to caffeine (Pincheira et al., 1988) and D N A cross-linking agents such as mitomycin C, diepoxybutane, nitrogen mustard and cisplatin (see Auerbach et al., 1989). The growth capacity of FA lymphoblasts is strongly reduced by those mutagens (Ishida and Buchwald, 1982) as well as by psoralen monoadducts (Averbeck et al., 1988). The induction of sister-chromatid exchange that is normally induced by bifunctional agents is also strongly suppressed in FA cells (Latt et al., 1975). Recent studies of mutagen sensitive Drosophila strains have revealed a striking parallel between FA and the mus308 mutants of that organism (Boyd et al., 1990). In both the Drosophila and human disorders the cellular phenotype is characterized by selective hypersensitivity to D N A cross-linking agents and by elevated frequencies of spontaneous chromosomal aberrations. In addition, mutant mus308 °2 cells of Drosophila fail to recover semiconservative D N A synthesis following mutagen treatment (Brown and Boyd, 1981) as do ceils from the A complementation group of FA (Moustacchi et al., 1987). Biochemical and genetic studies of the mus308 mutants have revealed an enzyme alteration that is revealed as an increased p I (Boyd et al., 1990; Sakaguchi et al., 1990). This report documents an analogous defect in cells assigned to the A complementation group of FA. Materials and methods
Cell lines and cell culture. Lymphoblast fines from normal individuals and patients with FA
were derived as described (Ishida and Buchwald, 1982). SV40-transformed fibroblasts from a normal individual (GM00637D) and from a patient with FA (GM06914A) were purchased from the Human Genetic Mutant Cell Repository (Camden, N J). The FA cell line GM06914A has been assigned to the A complementation group (Duckworth-Rysiecki et al., 1986). The control lymphoblast line K562 (GM512) was obtained from the Human Genetic Mutant Cell Repository and the fibroblast line AVneo from E.C. Friedberg. Lymphoblasts were cultured either in RPMI Media 1640 (Gibco) supplemented with 12% fetal bovine serum (Gibco) or in MEM supplemented with non-essential amino acids and 12% fetal bovine serum. Fibroblasts were maintained in alphaMEM with Hanks' salts (Gibco) supplemented with 15% fetal bovine serum. All cultures were maintained at 37°C in a humidified atmosphere containing 5% CO z. Culture medium was changed twice a week. Analysis of mutagen sensitivity. Sensitivity of selected lymphoblast cell lines to D N A crosslinking agents was monitored with a modification of the procedure described by Ishida and Buchwald (1982). Single-cell suspensions derived from exponentially growing cultures were distributed to 35 mm culture dishes at a density of 0.5 x 10 6 cells/ml. One day later the cells were treated with mutagen dissolved in physiological saline. 4 - 5 days after mutagen treatment metabolically active cells identified by dye exclusion were counted. Nuclease assay. The standard assay reaction of 200 /xl contained 150 /d of 50 m M acetate at pH 5.5, 5 mM MgC12, 1 mM DTT, 15% glycerol, 0.1% Triton X-100, 5 mM ATP and 5 /~g [3H]E. coli DNA (about 12 500 cpm/txg DNA). The reaction was initiated by addition of 50/xl of enzyme extract and terminated 3-5 h later by cooling and the addition of 200 t~l of 5 m g / m l bovine serum albumin and 0.6 ml of 10% ( w / v ) trichloroacetic acid. Each fraction was centrifuged and the radioactivity in the supernatant was determined by scintillation counting. The assay is linear with time between 0% and at least 50% solubilization of the substrate.
33
Organelle isolation. A particulate fraction was prepared from 1-3 x 107 cells hypotonically swollen for 10 rain in 5 ml of TE (10 mM Tris-HC1, pH 7.5, 1 mM EDTA). Following addition of 1 ml of 1.45 M sucrose in TE, the cells were homogenized with 10 strokes in a Dounce homogenizer (Kontes, B pestle). The homogenate was centrifuged at 1700 × g for 5 min and the resulting supernate was further spun at 2000 x g for 5 min. The pellet derived by centrifuging that supernate at 12 000 x g for 20 min was suspended in 1.5 ml lysis buffer (50 mM Tris-HC1, pH 7.4, 1 mM EDTA, 5 mM fl-mercaptoethanol, 15% glycerol, 0.8 M NaC1, 0.4% Triton X-100, 1 mM PMSF and 1/~g/ml each of pepstatin and leupeptin). Following sonication for 5 sec, the sample was again centrifuged at 1 2 0 0 0 × g for 20 min, and the supernatant was analyzed by isoelectric focusing. In a restricted number of cases organelles were recovered from a sucrose step gradient. Isoelectric focusing. The isoelectric focusing procedure has been described (Sakaguchi and Boyd, 1985). Focusing was performed in ll0-ml gradients of 5-40% sucrose containing 0.75% (v/v) carrier ampholyte (pH ranges; 3.5-10 or 5-8), 5% glycerol, 5 mM fl-mercaptoethanol, 0.4% Triton X-100 and 1/~g/ml each of leupeptin and pepstatin. The gradients were formed at 350 V for 40 h at 4°C and collected in 2-ml fractions. In selected experiments cells from mutant and control lines were mixed prior to analysis. Equivalent results were obtained when mixing was performed at each of these stages.
ously documented (Ishida and Buchwald, 1982), the identity of selected cell cultures was verified by determining sensitivity to nitrogen mustard and mitomycin C (Table 1). That analysis reveals a 2-fold increase in the sensitivity of HSC99 cells to both mutagens. Similarly the HSC72 cell line is 4-fold more sensitive to these agents. These sensitivities are approximately equivalent to those observed by Matsumoto et al. (1989) and thus serve to establish that a Fanconi anemia phenotype is being expressed under the experimental conditions employed in this study. The fact that these values are lower than those measured earlier by Ishida and Buchwald (1982) is probably attributable to the different criteria employed to assay inhibition of cell growth.
Identification of an organelle DNAase. Guided by previous studies of the model organism Drosophila (Boyd et al., 1990), biochemical analysis of these Fanconi lines has focused on a deoxyribonuclease derived from a particulate fraction composed predominantly of mitochondria and lysosomes. Preliminary characterization of that activity has established a pH optimum of 5.5 and a 4-fold preference for native over denatured DNA. The activity is insensitive to N-ethylmaleimide but is increased in the presence of magnesium and TABLE 1 SENSITIVITY OF LYMPHOBLAST CELL LINES TO NITROGEN MUSTARD (HN2) AND MITOCYCIN C (MMC) Mutagen
Results
Cell cultures. This study focuses principally on the three lymphoblast cell lines HSC72, HSC99 and HSC527, which were derived from unrelated individuals who have been identified with the A complementation group of FA (DuckworthRysiecki et al., 1985, 1986). Confirmatory results have also been obtained with the fibroblast line GM6914 that was derived from an effected sibling of the individual who gave rise to HSC527 (Duckworth-Rysiecki et al., 1986). Although hypersensitivity of the mutant cell lines to DNA cross-linking agents has been previ-
Cell line
Mutagen concentration Control
Fanconi group A
HSC93
HSC99
HSC72
HN2 (nM) Relative sensitivity
490+ 119 (6)
197+89 (5)
113+23 (3)
MMC(nM) Relative sensitivity
105+ 33(5)
1.0
1.0
2.5 54+15(4) 1.9
4.3 24+ 3(2) 4.4
The indicated mutagen concentrations reduce cell viability by 50% relative to untreated cultures. Those concentrations are presented with standard deviations and the number of determinations in brackets. Relative mutagen sensitivity is the control value divided by the corresponding control or mutant value.
34 A T P ( u n p u b l i s h e d observations). The p r i n c i p l e exp e r i m e n t a l p a r a m e t e r a s s a y e d in this s t u d y is the p I o f that enzyme. T h a t value is m o n i t o r e d b y i d e n t i f y i n g the e n z y m e p o s i t i o n in a p H g r a d i e n t following p r o t e i n f r a c t i o n a t i o n b y isoelectric focusing. T h e t o p p a n e l of Fig. 1 reveals the d i s t r i b u t i o n of nuclease activity in a p H g r a d i e n t e x t e n d i n g f r o m 5 to 8. T h e p e a k of activity rec o v e r e d f r o m n o r m a l cells occurs at a p I of a b o u t 6.3.
5
6
7 _--
~"
.P'4
8
::.~
GM637D
...............
GM6914
I
•
:: . . ::: ...
..............I ).............)
Aberrant p I in Fanconi anemia cells.
The center p a n e l of Fig. 1 reveals that the e n z y m e f r o m a F A - A f i b r o b l a s t cell line exhibits a p I that is a b o u t 0.5 units higher t h a n that of the control. T h e s h a d e d b a r on the left indicates that the range of the p e a k p I values o b s e r v e d for all a n a l y z e d c o n t r o l cell lines varies b e t w e e n 6.2 a n d 6.4. T h e s h a d e d b a r on the fight reveals that the p I values o b s e r v e d in the four a v a i l a b l e F A - A lines a n d their derivatives v a r y b e t w e e n 6.7 a n d 7.0. Since these two ranges d o n o t overlap, this p a r a m e t e r p r o v i d e s a reliable d i a g n o s t i c of the F A - A defect. A s shown in the b o t t o m p a n e l o f that figure, the u n i q u e i d e n t i t y of the c o n t r o l a n d F A e n z y m e s is e s t a b l i s h e d b y their s e p a r a t i o n o n a single focusing gradient. I n that e x p e r i m e n t cells f r o m the two cultures were m i x e d p r i o r to cell d i s r u p t i o n a n d e n z y m e fractionation. T h a t c o n t r o l excludes fract i o n a t i o n errors a n d reduces the l i k e l i h o o d that the o b s e r v e d p I difference is d u e to artifacts introd u c e d b y p r o t e o l y s i s or aggregation. T h e m i x i n g c o n t r o l also establishes that a n y m o d i f i c a t i o n factors, which might b e r e s p o n s i b l e for c o n v e r t i n g one e n z y m e f o r m to another, are n o t active u n d e r the c o n d i t i o n s e m p l o y e d for e n z y m e e x t r a c t i o n a n d f r a c t i o n a t i o n . If that were so, one e n z y m e f o r m w o u l d be c o n v e r t e d to the o t h e r d u r i n g f r a c t i o n a t i o n following cell mixing. D a t a in Fig. 2 e x t e n d the initial o b s e r v a t i o n s m a d e in f i b r o b l a s t s to a series of l y m p h o b l a s t cell lines. In that figure d a t a o b t a i n e d with two different c o n t r o l cell lines are p r e s e n t e d in the t o p p a n e l s for direct c o m p a r i s o n with d a t a f r o m the three available F A - A cell lines. I n each case the p I of the F A nuclease is r e p r o d u c i b l y higher t h a n that of the control. A n a l y s i s of m i x t u r e s of c o n t r o l a n d F A - A cells a g a i n c o n f i r m the u n i q u e i d e n t i t y of these two e n z y m e forms. A consistent shift in
I !:::::::::,::!
Mix
5
!::::::::::::!
:.:::::::::::" : : : " :
6 7 pH Within Gradient
8
Fig. 1. An organeller nuclease from an SV40 transformed FA fibroblast cell line exhibits an altered pl. A particulate fraction was prepared from control (GM00637D) and FA-A (GMO6914A) fibroblast cell lines. Protein was solubilized from the isolated organelles by sonication as described in Materials and Methods. Following centrifugation, the extracts were subjected to preparative isoelectric focusing and fractions were assayed for nuclease activity. In the bottom panel, an equal number of cells from those two lines were mixed prior to extraction. Nuclease activity in the peak fractions varied from 875 to 927 cpm solubilized per h. The shaded bar on the left designates the range of pl observed in the four different control cell lines assayed in this study (10 determinations). The shaded bar on the right represents the range of pI values found in the four FA-A lines assayed in this study (6 determinations).
p I has therefore been d o c u m e n t e d in two cell t y p e s d e r i v e d f r o m three d i f f e r e n t families affected with m u t a t i o n s in the F A - A gene.
35
The normal p I is expressed as a dominant trait. In both Fanconi anemia in man (DuckworthRysiecki et al., 1986) and in the mus308 mutants of Drosophila (Boyd et al., 1990) cellular hypersensitivity to D N A cross-linking agents is a recessive trait. In contrast, the wild-type form of the nuclease in Drosophila is dominant (Sakaguchi et al., 1990). That result indicates that the Drosophila gene is indirectly responsible for alteration of the nuclease p l rather than directly encoding a nuclease polypeptide. The question of dominance has been investigated in the FA-A disorder with a tetraploid cell line derived by cell fusion. That line was previously generated in complementation studies of FA (Duckworth-Rysiecki et al., 1985, 1986). D a t a presented in Fig. 3 reveal that a cell line generated by fusing the FA-A cell line HSC72 with the control lymphoblast line HSC93 expresses the control pattern of activity. This result suggests that the normal p l is dominant and 5
6
7
85
6
7
85
6
6
7
85
6
7
85
6
7
iiiiiii I000 "
~
iiiiiiii 500" ;:::;;:::
61 4
iiiiiiiii ~i!ii'i , 5 6 7 pH within gradient
8
Fig. 3. Nuclease analysis following cell fusion, A tetraploid cell line generated by fusion of the FA-A cell line HSC72 and the control cell line HSC93 was analyzed by isoelectric focusing as described in Fig. I.
thereby adds support to the hypothesis that the FA gene is required for the nuclease to assume the lower p I form.
Discussion
7
"r.
5
1500
8
pH within gradient Fig. 2. Alteration of the nuclease pI in lymphoblast cell lines of FA-A cultures. Nuclease was fractionated by isoeleetric focusing and assayed as described in Fig. 1. Control patterns are presented along the top row for comparison. The control data generated with K562 ceUswas obtained with a particulate fraction recovered from a discontinuous sucrose gradient. The mixing experiments presented in the lower panels were performed by combining an equal number of cells from the two cultures depicted above in each case. As in Fig. 1, the shaded bar on the left designates the range of pl observed in four different control cell lines and that on the right represents the range of p l values determined for all FA-A lines. Nuclease activity in the peak fractions varied from 1455 to 3375 c p m solubilized per h.
This study provides a direct association between the modification of an organelle nuclease activity and the A complementation group of FA. The consistency of the control p I observed in several different cell types strengthens the generality of the control observation. An increase in p I has been observed in one fibroblast cell line and three lymphoblast lines derived from FA-A patients. The results of the mixing experiments support the supposition that the different enzyme forms have unique identities and are not due to artifacts g e n e r a t e d d u r i n g p r e p a r a t i o n or fractionation of the enzyme. Since two of the individuals donating these cells are siblings (Duckworth-Rysiecki et al., 1986), this phenotype has been documented for three independently investigated families. Cell fusion studies have also identified two cell lines that fall outside of the A complementation group of FA (DuckworthRysiecki et al., 1985). A parallel analysis of the nuclease in those lines has been initiated (Sakaguchi, unpublished), but an understanding of the results must await the completion of complementation tests that are currently being conducted. The hypothesis that the FA-A gene is required for modification of the nuclease p I is supported
36
by parallel studies of the mus308 mutants of Drosophila. Those mutants, whose study motivated this investigation, are analogous to FA-A in that they exhibit hypersensitivity to D N A cross linking agents without expressing a strong sensitivity to monofunctional alkylating agents (Boyd et al., 1990). Like FA-A cells, the Drosophila mutants also exhibit an elevated level of spontaneous chromosome aberrations and a failure to recover D N A synthesis following mutagen treatment (see Schroeder-Kurth et al., 1989; Gatti et al., 1984; Brown and Boyd, 1981). At least four independently derived mutations at that locus result in a shift in nuclease p I that is analogous to that reported here for FA-A (Boyd et al., 1990). Because independent structural gene mutations are unlikely to produce precisely the same alteration in protein charge, it is probable that these mutations in both organisms are responsible for altering the p I of a nuclease polypeptide. Our demonstration that the normal phenotype is dominant in fusions between FA-A and normal cells (Fig. 3, Table 1) is consistent with that hypothesis. If the FA-A gene were to directly encode a polypeptide of the nuclease, codominant expression rather than dominance would be expected in that situation. Attempts are therefore being made to identify in vitro conditions that will permit conversion of the mutant enzyme form to that of the control cells in an effort to identify the postulated modification factor. Preliminary studies suggest that the two activities may be differentially phosphorylated. A potentially homologous enzyme has also been analyzed in nine recently isolated Chinese hamster cell lines that are hypersensitive to DNA crosslinking agents (Zdzienicka et al., in press). Two of those lines have been shown to exhibit an enzyme modification analogous to that observed in the Drosophila and FA cells (unpublished; this report). Observations of an analogous nuclease defect in cross-link sensitive mutants in a third organism further strengthens the correlation between that phenotype and the aberrant nuclease p l reported here. That result, therefore, further increases the rationale for investigation of the altered nuclease p l in FA-A. This study focuses on a nuclease that is recovered from a particulate fraction with sedimentation characteristics of mitochondria and lyso-
somes. Electron microscopic observations (Singson, unpublished observations) have confirmed the prevalence of mitochondria in that fraction. An apparently analogous Drosophila enzyme has been shown by differential fractionation of organelles on continuous Ficoll gradients to be associated specifically with mitochondria (Sakaguchi et al., 1990). Further studies are needed, however, to establish whether mitochondria are the exclusive repository of this enzyme form in either Drosophila or man. Given that the enzyme is at least partially associated with subcellular organelles, it is appropriate to consider whether it can be identified with any previously studied organeller nucleases. Although a definitive comparison will depend upon current efforts to purify and characterize this enzyme, present evidence fails to establish a clear analogy with any previously isolated mammalian enzyme. It is probably not the classical lysosomal acid-active nuclease (Yamanaka et al., 1974), because it shares many properties with a Drosophila enzyme that is present in mutants lacking the putative lysosomal activity (Boyd et al., 1990). Properties of available crude preparations also do not correspond to those of two mitochondrial nucleases that have recently been characterized. A mitochondrial activity recovered from mouse cells by Tompkinson and Linn (1986) has no activity on native DNA, which is the substrate utilized in this study. Likewise a mitochondrial nuclease isolated from bovine, rat and man (Cummings et al., 1987; Low et al., 1988) is active at pH 7 - 8 whereas the enzyme under study here is not. It is, therefore, possible that the nuclease perturbed in FA cells represents a new enzyme form, although contaminants in current preparations could be obscuring its relationship to previously identified enzymes. Similarities between the phenotype of the mus308 mutants in Drosophila and FA in man prompted the current investigation which has further extended that analogy through observations of a nuclease modification in FA-A cells. Identification of a potentially common function in these two diverse multicellular eukaryotes opens the possibility for a genetic analysis of the Fanconi A function in Drosophila. In an effort to exploit that opportunity, efforts are currently underway to clone and characterize the Drosophila mus308
37 gene. T h a t a p p r o a c h will u l t i m a t e l y p e r m i t a deftnitive definition of the relationship between that g e n e a n d t h e F A - A gene.
Acknowledgements W e are g r a t e f u l to D r s . A. G a n e s a n , C. L a m b e r t a n d M . A . D i e c k m a n n for s u p p l y i n g cell cultures. D r s . I.R. L e h m a n a n d W. S i e d e p r o v i d e d h e l p f u l discussions. D r . E . C . F r i e d b e r g g e n e r o u s l y provided resources and encouragement during a s a b b a t i c a l l e a v e o f J.B.B. in his l a b o r a t o r y . F i n a n cial s u p p o r t w a s p r o v i d e d b y t h e U . S . D e p a r t m e n t o f E n e r g y (EV70210), t h e U n i t e d S t a t e s P u b l i c Health Service (GM32040), The Fanconi Anemia R e s e a r c h F u n d , Inc. a n d the M e d i c a l R e s e a r c h Council of Canada (MT7220).
References Auerbach, A.D., R. Ghosh, P.C. Pollio and M. Zhang (1989) Diepoxybutane test for prenatal and postnatal diagnosis of Fanconi anemia, in: Fanconi Anemia: Clinical, Cytogenetic and Experimental Aspects, Springer, Berlin, pp. 71-82. Averbeck, D., D. Papadopoulo and E. Moustacchi (1988) Repair of 4,5',8-Trimethylpsoralen plus light-induced DNA damage in normal and Fanconi's anemia cell lines. Cancer Res., 48, 2015-2020. Boyd, J.B., K. Sakaguchi and P.V. Harris (1990) The mus308 mutants of Drosophila exhibit hypersensitivity to DNA cross-linking agents and are defective in a deoxyribonuclease, Genetics, 125, 813-819. Brown, T.C., and J.B. Boyd (1981) Abnormal recovery of DNA replication in ultraviolet-irradiated cell cultures of Drosophila melanogaster which are defective in DNA repair, Mol. Gen. Genet., 183, 363-368. Cummings, O.W., T.C. King, J.A. Holden and R.L. Low (1987) Purification and characterization of the potent endonuclease in extracts of bovine heart mitochondria, J. Biol. Chem., 262, 2005-2015. Diatloff-Zito, C., D. Papadopoulo, D. Averbeck and E. Moustacchi (1986) Abnormal response to DNA crosslinking agents of Fanconi anemia fibroblasts can be corrected by transfection with normal human DNA, Proc. Nail. Acad. Sci. (U.S.A.), 83, 7034-7038. Digweed, M., and K. Sperling (1989) Identification of a HeLa mRNA fraction which can correct the DNA-repair defect in Fanconi anaemia fibroblasts, Mutation Res., 218, 171177. Duckworth-Rysiecki, G., C.A. Cornish, C.A. Clarke and M. Buchwald (1985) Identification of two complementation groups in Fanconi anemia, Somat. Cell Mol. Genet., 11, 35-41.
Duckworth-Rysiecki, G., L. Toji, J. Ng, C. Clarke and M. Buchwald (1986) Characterization of a Simian virus 40transformed Fanconi anemia fibroblast cell line, Mutation Res., 166, 207-214. Gatti, M., S. Pimpinelli, C. Bove, B.C. Baker, D.A. Smith, A.T.C. Carpenter and P. Ripoll (1984) Genetic control of mitotic cell division in Drosophila melanogaster, in: Genetics: New Frontiers, Oxford and IBS Publishing Co., New Delhi, pp. 193-204. GiSk, M.M., and E. Wunder (1987) Microinjection of normal cell extracts into Fanconi anemia fibroblasts corrects defective scheduled DNA synthesis recovery after 8-methoxypsoralen plus UVa treatment, Human Genet., 75, 350-355. Ishida, R., and M. Buchwald (1982) Susceptibility of Fanconi's anemia lymphoblasts to DNA-cross-linking and alkylating agents, Cancer Res., 42, 4000-4006. Latt, S.A., G. Stetten, L.A. Juergens, G.R. Buchanan and P.S. Gerald (1975) Induction by alkylating agents of sister chromatid exchanges and chromatid breaks in Fanconi's anemia, Proc. Natl. Acad. Sci. (U.S.A.), 72, 4066-4070. Low, R.L., J.M. Buzan and C.L. Couper (1988) The preference of the mitochondrial endonuclease for a conserved sequence block in mitochondrial DNA is highly conserved during mammalian evolution, Nucl. Acids Res., 16, 64276445. Matsumoto, A., J.-M.H. Vos and P.C. Hanawalt (1989) Repair analysis of mitomycin C-induced DNA crosslinking in ribosomal RNA genes in lymphoblastoid cells from Fanconi's anemia patients, Mutation Res., 217, 185-192. Moustacchi, E., D. Papadopoulo, C. Diatloff-Zito and M. Buchwald (1987) Two complementation groups of Fanconi's anemia differ in their phenotypic response to a DNA-crosslinking treatment, Human Genet., 75, 45-47. Pincheira, J., M. Bravo and J.F. Lopez-Saez (1988) Fanconi's anemia lymphocytes: effect of caffeine, adenosine and niacinamide during G 2 prophase, Mutation Res., 199, 159165. Sakagnchi, K., and J.B. Boyd (1985) Purification and characterization of a DNA polymerase fl from Drosophila, J. Biol. Chem., 260, 10406-10411. Sakaguchi, K., P.V. Harris, R. van Kuyk, A. Singson and J.B. Boyd (1990) A mitochondrial nuclease is modified in Drosophila mutants (mus308) that are hypersensitive to DNA crosslinking agents, Mol. Gen. Genet., 224, 333-340. Schroeder, T.M. (1982) Genetically determined chromosome instability syndromes, Cytogenet. Cell. Genet., 33, 119-132. Schroeder-Kurth, T.M., A.D. Auerbach and G. Obe (1989) Fanconi anemia: Clinical, Cytogenetic and Experimental Aspects, Springer, Berlin. Shaham, M., B. Adler, S. Ganguly and R.S.K. Chaganti (1987) Transfection of normal human and Chinese hamster DNA corrects diepoxybutane-induced chromosomal hypersensitivity of Fanconi anemia fibroblasts, Proc. Natl. Acad. Sci. (U.S.A.), 84, 5853-5857. Tompkinson, A.E., and S. Lima (1986) Purfication and properties of a single strand-specific endonuclease from mouse cell mitochondria, Nucleic Acid. Res., 14, 9579-9593.
38 Yamanaka, M., Y. Tsubota, M. Anai, K. Ishimatsu, M. Okumura, S. Katsuki and Y. Takagi (1974) Purification and properties of acid deoxyribonucleases of human gastric mucosa and cervix uteri, J. Biol. Chem., 249, 3884-3889. Zakrzewski, S., and K. Sperling (1980) Genetic heterogeneity
of Fanconi's anemia demonstrated by somatic cell hybrids, Hum. Genet., 56, 81-84. Zdzienicka, M.Z., J.W.I.M. Simons and P.H.M. Lohman (1990) DNA Repair Mechanisms and Their Biological Implications In Mammalian Cells, Plenum, New York, in press.
