Mutation Research, 27 (1975) 407-409

© Elsevier Scientific Publishing Company, Amsterdam---Printed in The Netherlands

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HUMAN CELLS R E P A I R DNA DAMAGED BY N I T R O U S ACID

RUFUS S. DAY, III Chemistry Branch, DCCP, NCI, National Institutes of Health, Bethesda, Md. 2OOl4 (U.S.A .)

(Received August 2oth, 1974) (Revision received November 8th, 1974)

SUMMARY Cultured fibroblasts from normal persons or persons afflicted b y xeroderma pigmentosum were used as hosts for adenovirus 2 infection. With xeroderma cells as hosts, nitrous acid-treated virus showed less plaque-forming ability than when normal cells were used, indicating that DNA damaged by nitrous acid is at least partly repaired by normal human cells.

The development of a sensitive assay for repair of UV-damaged DNA by human cells using irradiated adenovirus 2 as an intraeellular repair substrate 8 prompted me to investigate the ability of human cells to repair damage caused by other agents. One such agent, nitrous acid, was chosen for study after consideration of the following: it gives rise to a high number of point mutations11,15 in bacterial, phage, and plant viral systems, as well as to large deletion mutations in T 4 phage of E. col#*. In the case ot E. coli phage SI 3, nitrous acid production of all possible base transitions (G -+ A, A -+ G, C --~ T, T -.~ C) have been detected genetically 16. Serratia phage kappa appears 5-20% more sensitive to nitrous acid inactivation when using UV-sensitive hosts than when using wild-type bacteria as hosts 17. Moreover, E. coli mutants having reduced ability to excise UV-produced pyrimidine dimers are more sensitive (as measured by colony-fornfing ability) to the lethal effects of nitrous acid than their wild-type parent strains 9 and also more mutable by nitrous acid 4. Similarly, some but not all UV-sensitive mutants of several eukaryotic organisms including Neurospora crassa 3, Aspergillus rugulosus 1°, and Saccharomyces cerevisiae 18 have also been reported to be sensitive to nitrous acid killing. The DNA changes known to be produced b y nitrous acid (and presumed to be responsible for some of the above effects), include the deamination of adenine, guanine, and cytosinen, ~3 (-NH2 replaced b y -OH in all cases) as well as crosslinkages of, as yet, undefined structure s. To test if nitrous acid damage is repaired by human cells, we added o.I ml adenovirus 2 stock s containing about 3" lO7 plaque forming units (pfu) per ml to 1.8 inl 0.25 M sodiunl acetate buffer, p H 5.0, at 37 °, and then added 0.6 ml I M sodium nitrite, also at 37 ° , to start nitrous acid treatment. Samples taken during the treatAbbreviation: XP, xeroderma pigmentosum.

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ment period were diluted I:IOO in basal medium Eagle with 1% fetal calf serum to stop the reaction. The control was treated with buffer but not sodium nitrite. The treated viral suspensions were titered on monolayers of cells having either normal or reduced ability to repair UV-damaged DNA using a previously published I7-day plaque assay 8. (Normal strains KD and ND, and xeroderma pigmentosum (XP) strains X P I L O and X P I 2 B E (referred to previously as 12Ol and 1223 respectively) have been describedS, 7. Normal human fibroblast strain HG8oo was the kind gift of Dr. James German, New York Blood Center. The X P 4 B E strain was developed from a biopsy from XP patient 4 of BURK et al. 2 referred to previously as XW6, 7, and shows 70% of normal repair of UV-damaged adenovirus 2 (ref. 7) but has been estimated to have normal UV repair by others1,*,5.) Virus treated for 1 or 30 min with buffer only gave 1.o-1.3" lO 6 pfu/ml buffer on all cell strains tested, as did virus treated with no buffer at all. The plaque forming ability of nitrous acid treated virus, however, depended upon the cell strain used for plaquing (Fig. I). When the normal strains were used as hosts the viral inactivation slope was about 0.5 of that measured when the XP strains X P I L O and X P I 2 B E were used. We therefore believe that the number of plaques due to treated virus as measured using normal or X P 4 B E cell monolayers is conceptually divisible into two populations: That (measured using X P strains X P I L O or X P I 2 B E monolayers) which is made in the absence of the repair lacking in these cells, and that made due to reactivation of damaged virus by the repair process in the normal cells. These particular experiments have been done twice with similar results. 100

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4 8 12 16 20 MINUTES NITROUSACID TREATMENT

Fig. i. N i t r o u s acid i n a c t i v a t i o n of a d e n o v i r u s 2. &, K D ; A, H G 8 o o ; I , N D ; [], X P 4 B E ;

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X P I L O ; O, X P I 2 B E .

The X P strains X P I L O and X P I 2 B E used here belong to X P gene complementation group A TM. The use oI X P strains belonging to groups C and D resulted in inactivation slopes falling between tile slopes measured on normal and X P group A strains (not shown). Evidently UV-sensitive human as well as Serratia cells 17 show a deiect in tile repair of nitrous acid treated viruses. The difference in response of X P and normal cells to nitrous acid damaged virus is rather small (about two-fold) in comparison to

REPAIR OF NITROUS ACID DAMAGE IN D N A

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t h a t o b s e r v e d u s i n g U V - i r r a d i a t e d v i r u s w i t h t h e s a m e cells (14- t o 25-fold). T h i s m a y b e b e c a u s e e i t h e r (z) X P cells a r e c o m p l e t e l y d e f i c i e n t i n r e p a i r of n i t r o u s a c i d d a m a g e w h i l e n o r m a l cells a r e o n l y p a r t l y c a p a b l e of s u c h r e p a i r ( p o s s i b l y b e c a u s e of d a m a g e i n a c r i t i c a l n o n - D N A c o m p o n e n t ) , or (2) b o t h n o r m a l a n d X P cells r e p a i r n i t r o u s a c i d d a m a g e r e l a t i v e l y well, b u t X P cells a r e c a p a b l e of o n l y h a l f n o r m a l r e p a i r . ACKNOWLEDGEMENTS I t h a n k D r s . C. WESLEY DINGMAN, KENNETH H . KRAEMER, a n d JAY H . ROBBINS f o r a d v i c e , h e l p f u l d i s c u s s i o n s , a n d r e a d i n g t h e m a n u s c r i p t , a n d Mrs. BARBARA HEIFETZ for e x p e r t a s s i s t a n c e w i t h t h e m a n u s c r i p t . REFERENCES I BURK, P. G., M. A. LUTZNER, D. D. CLARKE AND J. H. ROBBINS, Ultraviolet-stimulated

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thymidine incorporation in xeroderma pigmentosum lymphocytes, J. Lab. Clin. Med., 77 (1971 ) 759-767 . BURK, P. G., S. I-I. YUSPA, M. A. LUTZNER AND J. H. ROBBINS, Xeroderma and DNA repair, Lancet, i (1971) 6Ol. CHANG, L. T., AND R. W. TUVESON, Ultraviolet-sensitive m u t a n t s in Neurospora crassa, Genetics, 56 (1967) 8Ol-81o. CLARKE, C. H., Repair systems and nitrous acid mutagenesis ill E. coli B/r, Mutation Res., 9 (197 ° ) 359-368. CLEAVER, J. E., DNA damage and repair in light-sensitive h u m a n skin disease, J. Invest. Dermatol., 54 (197 °) 181-195. DAY', R. S., 111, Cellular reactivation of ultraviolet-irradiated h u m a n adenovirus 2 in normal and xeroderma pigmentosum fibroblasts, Photochem. -Photobiol., 19 (1974) 9-13. DAY, R. S., 111, Studies on repair of adenovirus 2 using normal, xeroderma pigmentosum, and X P heterozygous strains, Cancer Res., 34 (1974) 1965-1969. GEIDUSCHEK, E. P., Reversible DNA, Proc. Natl. Acad. Sci. (U.S.), 47 (1961) 950-955 . HOWARD-FLANDERS, P., AND R. P. BOYCE, DNA repair and genetic recombination: studies on m u t a n t s of Escherichia coli defective in these processes, Radiation Res., Suppl. 6 (1966) 156-181. LENNOX, J. E., AND R. W. TUVESON, The isolation of ultraviolet-sensitive m u t a n t s from AspergiUus rugulosus, Radiation Res., 31 (1967) 382-388. ORGEL, L. E., The chemical basis of mutation, Adv. Enzymol., 27 (1965) 289-346. ROBBINS, J. H., K. H. KRAEMER, M. A. LUTZNER, B. W. FESTOFF AND H. G. COON, Xeroderma pigmentosum, an inherited disease with sun sensitivity, multiple cutaneous neoplasms, and abnormal DNA repair. Ann. Internal Med., 8o (1974) 221-248SINGER, B., AND H. FRAENKEL-CONRAT, The role of conformation in chemical mutagenesis, Progr. Nucleic Acid Res. Mol. Biol., 9 (1969) 1-29. TESSMAN, I., The induction of large deletions by nitrous acid, J. Mol. Biol., 5 (1962) 442-445 . TESSMAN, I., R. K. PODDAR AND S. KUMAR, Identification of the altered bases in m u t a t e d single-stranded DNA, I. In vitro mutagenesis by hydroxylamine, ethyl methanesulfonate and nitrous acid, J. Mol. Biol., 9 (1964) 352-363 • VANDERBILT, A. S., AND I. TESSMAN, Identification of the altered bases in m u t a t e d singlestranded DNA, IV. Nitrous acid induction of the transitions guanine to adenine and thymine to cytosine, Genetics, 66 (197 o) i - i o . WINKLER, W., Wirtzellreaktivierung chemich induzierter letalschaden im DNA-haltigen Serratia-phagen kappa, Z. Naturforsch., 2ob (1965) 864-867. ZIMMERMAN, F. K., Sensitivity to methyl methanesulfonate and nitrous acid of ultraviolet light-sensitive m u t a n t s in Saccharomyces cerevisiae, Mol. Gen. Genet., lO2 (1968) 247-256.

Human cells repair DNA damaged by nitrous acid.

Cultured fibroblasts from normal persons or persons afflicted by xeroderma pigmentosum were used as hosts for adenovirus 2 infection. With xeroderma c...
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