- Email: [email protected]
Ataxia telangiectasia: Further considerations of the evidence for single strand break repair
415
Mutation Research, 61 (1979) 415--417 © Elsevier/North-Holland Biomedical Press
Short Communication ATAXIA TELANGIECTASIA: FURTHER CONSIDERATIONS OF THE EVIDENCE FOR SINGLE STRAND BREAK REPAIR
R.B. SHERIDAN III and P.C. HUANG *
Department of Biochemistry, The Johns Hopkins University, School of Hygiene and Public Health, Baltimore, MD 21205 (U.S.A.) (Received 21 November 1978) (Revision rbceived 13 February 1979) (Accepted 27 February 1979)
Fibroblast cells derived from 6 Ataxia lines were compared with normal cells in their ability to rejoin single-strand DNA breaks caused by ionizing radiation. In a newly developed enzymatic assay, the Ataxia cells show extensive ligation of the breaks within 90 min of repair incubation. Ataxia telangiectasia (AT) is a human autosomal recessive disease characterized by neurodegeneration, oculocutaneous telangiectasias, variable immunological defects, predisposition towards malignancy and extreme sensitivity to ionizing radiations [1]. Cells derived from patients with AT have displayed a higher than normal frequency of chromatid aberrations following GI irradiation [4,7]. This observation led cytogeneticists to propose that ataxia is deficient in the repair of single'-strand breaks in DNA [7]. Since then a large a m o u n t of biochemical evidence has been accumulated demonstrating that AT is strand-break repair-proficient [2--6,9]. However, a new report based on cytogenetic evidence suggesting that "the basic defect in all AT patients may be an inability to repair all DNA strand breaks ", has recently appeared [8]. Concerned with the possibility that alkaline sucrose gradients were not sensitive enough to detect a small difference in the repair capabilities among the various cell lines tested, we developed a more sensitive m e t h o d based on the rate of DNA-strand separation in alkali [5]. This technique was shown to be sensitive enough to detect at least one single-strand break in 109 dalton DNA, while repair of strand scissions could be quantitated at doses of radiation below 400 rad. In order to resolve the conflict between cytogenetic versus other biochemical evidence, we have extended our earlier work on AT by measuring the capacity for 6 ataxia fibroblast cell lines to repair single-strand breaks induced * To whom
correspondence should be addressed.
416
in DNA by 400 tad of radiation. The results are summarized in Table 1. There is no apparent difference in either the rate or extent of repair between all 6 ataxia cell lines and the normal control. Thus the generalized notion that all AT cell lines are deficient in rejoining DNA-strand breaks cannot be supported even at very low doses of radiation. In addition to the cytogenetic evidence, the strand-break repair deficiency argument [8] rests on interpretation of studies in which 6 ataxia lines were found to demonstrate reduced repair-replication [7]. Two of these lines (CRL 1312, CRL 1343) have been examined here for strand-break repair after low dose irradiation and are found to be normal. Previously, CRL 1312 and another line (AT 3BI) were reported to be deficient in the incision step near 7-radiation induced damages in DNA. Thus reduced repair-replication has already been shown to be linked to a deficiency in incision not strand-break repair. Of the cell lines used in this recent study [8], AT 4BI, AT 5BI, and AT 7BI were shown to be normal with respect to repair replication and one AT 4BI may be incision-proficient [3]. From the data presented in Table 1, we now know that AT 4BI and AT 5BI also repair-strand breaks normally. It would be interesting to see if any differences in aberration production could be detected between incision-defective lines like CRL 1312 and AT 3BI and the repair-replication normal lines AT 4BI, AT 5BI and AT 7BI. Unfortunately, no distinction between these two classes of cell lines has y e t been made. It is conceivable that those AT lines exhibiting repair-replication abnormalities are incision-defective rather than strand-break repair or excision-deficient. All of the biochemical evidence published to date, including this work, does not support deficiency in the rejoining capacity for strand breakage. Even with the improved technique as used here, however, the sensitivity of measurement
TABLE 1 ATAXIA TELANGIECTASIA SINGLE-STRAND BREAKS
CELL LINES SCREENED
FOR
D E F I C I E N C Y IN T H E R E P A I R O F
F i b r o b l a s t s were s e e d e d at 5 × 104 cells/flask a n d labelled for 18 h, w i t h [ 3 H ] t h y m i d i n e (0.1 p C i / m l ) . E a c h g r o u p of 3 flasks was ~ - i r r a d i a t e d at a dose of 4 0 0 rad f o l l o w e d b y t r e a t m e n t with alkali for 30 rain a f t e r a p p r o p r i a t e r e p a i r intervals. U p o n n e u t r a l i z a t i o n , t h e f r a c t i o n o f d o u b l e - s t r a n d e d D N A r e m a i n i n g ( F ) was d e t e r m i n e d by r e s i s t a n c e to S 1 n u c l e a s e digestion. F r o m this d a t a , t h e p e r c e n t r e p a i r (R) was c o m p u t e d a n d c o r r e c t e d ( R c ) for the i n c r e a s e d d e v e l o p m e n t of alkali labile sites. See [5] for details. T h e 3 C R L lines w e r e o b t a i n e d f r o m t h e A m e r i c a n T y p e C u l t u r e C o l l e c t i o n a n d t h e 2 A T lines w e r e gifts f r o m Dr. A l a n L e h m a n n , MRC'- A T O 2 B A was originally d e s i g n a t e d VM a n d was e s t a b l i s h e d in this l a b o r a t o r y . Cell line
0 rain
15 rain
AT02BA AT4BI AT5BI CRL 1343 CRL 1347 CRL 1312
36.80 37,62 36,54 35,17 39.23 38,41
57.62 58.14 58.64 55.66 57.73 59.70
80.42 78.25 82.12 78.81 79.73 81.36
80.14 79.20 81.64 77.87 78.32 80.40
~7 S.D. R Rcc
37.20 1.32 0 0
57.92 1.22 48.75 48.80
80.12 1.35 101.20 101.54
79.60 1.28 100.00 I00.00
49.97
101.75
100.00
Unaffected control R e
0
90 m i n
Unirradiated control
417
cannot equate that detected by observing chromosomal aberrations. Therefore, at this juncture a direct correlation between aberration production and unrepaired strand break can at best be speculated but not demonstrated. In light of the negative results showing that AT is not necessarily a strand-break repair-deficiency syndrome, investigations into other possible modes of defect such as misrepair of breaks in phosphodiester bonds or failure of incision events near damaged nitrogenous bases which can now be tested may be in order. Acknowledgment Work supported by NIH grants (ROES1596, POES00454). References 1 K r a e m e r , K . H . , in: W.W. N i c h o l s a n d D. M u r p h y ( E d s . ) , D N A Repair Processes: Cellular Senescence a n d S o m a t i c Cell G e n e t i c s , S y m p o s i u m S p e c i a l i s t s , M i a m i , F l o r i d a , 1 9 7 7 , p p . 37---41. 2 L e h m a n n , A . R . , a n d S. Stevens, Biochim. B i o p h y s . A c t a , 4 7 4 ( 1 9 7 7 ) 4 9 - - 6 0 . 3 P a t e r s o n , M.C., B.P. S m i t h , D . A . K n i g h t , a n d A . K . A n d e r s o n , in: J. C a s t e l l a n i ( E d . ) , R e s e a r c h in Photobiology, Plenum, New York, 1 9 7 7 , pp. 2 0 7 - - 2 1 8 . 4 R a r y , J . M . , M . A . B e n d e r , a n d T.E. K e l l e y , A m . J. H u m a n G e n e t . , 26 ( 1 9 7 4 ) 7 0 A . 5 S h e r i d a n III, R . B . , a n d P.C. H u a n g , Nucl. A c i d s Res., 4 ( 1 9 7 7 ) 2 9 9 - - 3 1 8 . 6 T a y l o r , A . M . R . , D . G . H a r n d e n , C . F . A r l e t t , S.A. H a r c o u r t , A . R . L e h m a n n , S. S t e v e n s a n d B.A. Bridges, Nature (London), 258 (1975) 427--429. 7 Taylor, A.M.R., J.A. Metcalfe, J.M. Oxford and D.G. Harnden, Nature (London), 260 (1976) 441-443. 8 T a y l o r , A . M . R . , M u t a t i o n R e s . , 50 ( 1 9 7 8 ) 4 0 7 - - 4 1 8 . 9 V i n c e n t Jr., R . A . , R . B . S h e r i d a n III a n d P.C. H u a n g , M u t a t i o n R e s . , 3 3 ( 1 9 7 5 ) 3 5 7 - - 3 6 6 .
Mutation Research, 61 (1979) 415--417 © Elsevier/North-Holland Biomedical Press
Short Communication ATAXIA TELANGIECTASIA: FURTHER CONSIDERATIONS OF THE EVIDENCE FOR SINGLE STRAND BREAK REPAIR
R.B. SHERIDAN III and P.C. HUANG *
Department of Biochemistry, The Johns Hopkins University, School of Hygiene and Public Health, Baltimore, MD 21205 (U.S.A.) (Received 21 November 1978) (Revision rbceived 13 February 1979) (Accepted 27 February 1979)
Fibroblast cells derived from 6 Ataxia lines were compared with normal cells in their ability to rejoin single-strand DNA breaks caused by ionizing radiation. In a newly developed enzymatic assay, the Ataxia cells show extensive ligation of the breaks within 90 min of repair incubation. Ataxia telangiectasia (AT) is a human autosomal recessive disease characterized by neurodegeneration, oculocutaneous telangiectasias, variable immunological defects, predisposition towards malignancy and extreme sensitivity to ionizing radiations [1]. Cells derived from patients with AT have displayed a higher than normal frequency of chromatid aberrations following GI irradiation [4,7]. This observation led cytogeneticists to propose that ataxia is deficient in the repair of single'-strand breaks in DNA [7]. Since then a large a m o u n t of biochemical evidence has been accumulated demonstrating that AT is strand-break repair-proficient [2--6,9]. However, a new report based on cytogenetic evidence suggesting that "the basic defect in all AT patients may be an inability to repair all DNA strand breaks ", has recently appeared [8]. Concerned with the possibility that alkaline sucrose gradients were not sensitive enough to detect a small difference in the repair capabilities among the various cell lines tested, we developed a more sensitive m e t h o d based on the rate of DNA-strand separation in alkali [5]. This technique was shown to be sensitive enough to detect at least one single-strand break in 109 dalton DNA, while repair of strand scissions could be quantitated at doses of radiation below 400 rad. In order to resolve the conflict between cytogenetic versus other biochemical evidence, we have extended our earlier work on AT by measuring the capacity for 6 ataxia fibroblast cell lines to repair single-strand breaks induced * To whom
correspondence should be addressed.
416
in DNA by 400 tad of radiation. The results are summarized in Table 1. There is no apparent difference in either the rate or extent of repair between all 6 ataxia cell lines and the normal control. Thus the generalized notion that all AT cell lines are deficient in rejoining DNA-strand breaks cannot be supported even at very low doses of radiation. In addition to the cytogenetic evidence, the strand-break repair deficiency argument [8] rests on interpretation of studies in which 6 ataxia lines were found to demonstrate reduced repair-replication [7]. Two of these lines (CRL 1312, CRL 1343) have been examined here for strand-break repair after low dose irradiation and are found to be normal. Previously, CRL 1312 and another line (AT 3BI) were reported to be deficient in the incision step near 7-radiation induced damages in DNA. Thus reduced repair-replication has already been shown to be linked to a deficiency in incision not strand-break repair. Of the cell lines used in this recent study [8], AT 4BI, AT 5BI, and AT 7BI were shown to be normal with respect to repair replication and one AT 4BI may be incision-proficient [3]. From the data presented in Table 1, we now know that AT 4BI and AT 5BI also repair-strand breaks normally. It would be interesting to see if any differences in aberration production could be detected between incision-defective lines like CRL 1312 and AT 3BI and the repair-replication normal lines AT 4BI, AT 5BI and AT 7BI. Unfortunately, no distinction between these two classes of cell lines has y e t been made. It is conceivable that those AT lines exhibiting repair-replication abnormalities are incision-defective rather than strand-break repair or excision-deficient. All of the biochemical evidence published to date, including this work, does not support deficiency in the rejoining capacity for strand breakage. Even with the improved technique as used here, however, the sensitivity of measurement
TABLE 1 ATAXIA TELANGIECTASIA SINGLE-STRAND BREAKS
CELL LINES SCREENED
FOR
D E F I C I E N C Y IN T H E R E P A I R O F
F i b r o b l a s t s were s e e d e d at 5 × 104 cells/flask a n d labelled for 18 h, w i t h [ 3 H ] t h y m i d i n e (0.1 p C i / m l ) . E a c h g r o u p of 3 flasks was ~ - i r r a d i a t e d at a dose of 4 0 0 rad f o l l o w e d b y t r e a t m e n t with alkali for 30 rain a f t e r a p p r o p r i a t e r e p a i r intervals. U p o n n e u t r a l i z a t i o n , t h e f r a c t i o n o f d o u b l e - s t r a n d e d D N A r e m a i n i n g ( F ) was d e t e r m i n e d by r e s i s t a n c e to S 1 n u c l e a s e digestion. F r o m this d a t a , t h e p e r c e n t r e p a i r (R) was c o m p u t e d a n d c o r r e c t e d ( R c ) for the i n c r e a s e d d e v e l o p m e n t of alkali labile sites. See [5] for details. T h e 3 C R L lines w e r e o b t a i n e d f r o m t h e A m e r i c a n T y p e C u l t u r e C o l l e c t i o n a n d t h e 2 A T lines w e r e gifts f r o m Dr. A l a n L e h m a n n , MRC'- A T O 2 B A was originally d e s i g n a t e d VM a n d was e s t a b l i s h e d in this l a b o r a t o r y . Cell line
0 rain
15 rain
AT02BA AT4BI AT5BI CRL 1343 CRL 1347 CRL 1312
36.80 37,62 36,54 35,17 39.23 38,41
57.62 58.14 58.64 55.66 57.73 59.70
80.42 78.25 82.12 78.81 79.73 81.36
80.14 79.20 81.64 77.87 78.32 80.40
~7 S.D. R Rcc
37.20 1.32 0 0
57.92 1.22 48.75 48.80
80.12 1.35 101.20 101.54
79.60 1.28 100.00 I00.00
49.97
101.75
100.00
Unaffected control R e
0
90 m i n
Unirradiated control
417
cannot equate that detected by observing chromosomal aberrations. Therefore, at this juncture a direct correlation between aberration production and unrepaired strand break can at best be speculated but not demonstrated. In light of the negative results showing that AT is not necessarily a strand-break repair-deficiency syndrome, investigations into other possible modes of defect such as misrepair of breaks in phosphodiester bonds or failure of incision events near damaged nitrogenous bases which can now be tested may be in order. Acknowledgment Work supported by NIH grants (ROES1596, POES00454). References 1 K r a e m e r , K . H . , in: W.W. N i c h o l s a n d D. M u r p h y ( E d s . ) , D N A Repair Processes: Cellular Senescence a n d S o m a t i c Cell G e n e t i c s , S y m p o s i u m S p e c i a l i s t s , M i a m i , F l o r i d a , 1 9 7 7 , p p . 37---41. 2 L e h m a n n , A . R . , a n d S. Stevens, Biochim. B i o p h y s . A c t a , 4 7 4 ( 1 9 7 7 ) 4 9 - - 6 0 . 3 P a t e r s o n , M.C., B.P. S m i t h , D . A . K n i g h t , a n d A . K . A n d e r s o n , in: J. C a s t e l l a n i ( E d . ) , R e s e a r c h in Photobiology, Plenum, New York, 1 9 7 7 , pp. 2 0 7 - - 2 1 8 . 4 R a r y , J . M . , M . A . B e n d e r , a n d T.E. K e l l e y , A m . J. H u m a n G e n e t . , 26 ( 1 9 7 4 ) 7 0 A . 5 S h e r i d a n III, R . B . , a n d P.C. H u a n g , Nucl. A c i d s Res., 4 ( 1 9 7 7 ) 2 9 9 - - 3 1 8 . 6 T a y l o r , A . M . R . , D . G . H a r n d e n , C . F . A r l e t t , S.A. H a r c o u r t , A . R . L e h m a n n , S. S t e v e n s a n d B.A. Bridges, Nature (London), 258 (1975) 427--429. 7 Taylor, A.M.R., J.A. Metcalfe, J.M. Oxford and D.G. Harnden, Nature (London), 260 (1976) 441-443. 8 T a y l o r , A . M . R . , M u t a t i o n R e s . , 50 ( 1 9 7 8 ) 4 0 7 - - 4 1 8 . 9 V i n c e n t Jr., R . A . , R . B . S h e r i d a n III a n d P.C. H u a n g , M u t a t i o n R e s . , 3 3 ( 1 9 7 5 ) 3 5 7 - - 3 6 6 .