Biochemical basis of radiation-sensitivity in mutants of Neurospora crassa

WIutation Research, 19 (1973) 167-173 © Elsevier Scientific Publishing Company, A m s t e r d a m - Printed in The Netherlands

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BIOCHEMICAL BASIS OF R A D I A T I O N - S E N S I T I V I T Y IN MUTANTS OF CRASSA

NEUROSPORA

THOMAS E. W O R T H Y * AND J. L. E P L E R

Institute of Radiation Biology, University of Tennessee, Knoxville, and Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tenn. 3783 o (U.S.A.) (Received F e b r u a r y 2oth, 1973)

SUMMARY

The available UV-sensitive mutants of Neurospora crassa were examined for their ability to excise and photoreactivate cytosine-containing dimers in vivo. All strains exhibited in vivo photoreactivation, including upr-I, which was originally thought to be deficient in photoreactivation. Two strains, uvs-2 and upr-z were shown to be deficient in excision repair; uvs-3 was shown to contain a residual amount of excision capability. The remaining strains, uvs-I, uvs-5, and uvs-6, were normal in their ability to excise dimers. Based on these results, tentative analogies were drawn between the Neurospora mutants and the known classes of UV-sensitive mutants in E. coll. Accordingly, the N. crassa mutants were classified as uvs-x, -lon; uvs-2, -uvr; uvs-3, -uvr (rec?) ; uvs-5, -lon; uvs-6,-rec; and upr-z, -uvr. A comparison was made between the biochemical responses and the available published data on mutation induction in the Neurospora mutants. Although some relationships were seen between repair defects and mutation induction, too little data were available for any definitive conclusions.

INTRODUCTION

Mutants containing single gene defects at various loci have been extremely useful in the characterization of the series of enzymatic steps which, when taken together, form a unified biochemical pathway. This approach has had very limited success in the elucidation of the processes involved in the repair of radiation damage to the DNA of fungi because of the inability to examine the biochemical reactions involved in the various repair systems. With the advent of the requisite procedures (refs. 18, 2o), it is now possible to begin the necessary studies. Because of the success in characterizing radiation-sensitive mutants in bacterial systems such as Escherichia coli, it would be of great aid to be able to establish analogies between the available radiation-sensitive mutants of Neurospora crassa and other fungi and their bacterial counterparts whenever possible. * Present address: Division of R h e u m a t i c a n d Genetic Diseases, D e p a r t m e n t of Medicine, Duke University Medical Center, D u r h a m , N.C., 27706 (U.S.A.).

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T . E . WORTHY, j. L. EPLF_R

The isolation of UV-sensitive strains of E. coli has aided immensely in elucidating the various molecular events through which an organism can nullify the effects of exposure to UV. As a consequence of the isolation and characterization of such nmtants in the bacterial system, several distinct classes of UV sensitivity have been established: (i) mutants that are UV-sensitive and unable to reactivate UV-irradiated bacteriophage--HCR (host cell reactivation-deficient); (ii) strains that are sensitive to UV but are not HCR deficient -UVR (excision-repair deficient); (iii) strain,~ sensitive to both UV and ionizing radiation E X R ; (iv) strains sensitive to both UV and ionizing radiation and also deficient in recombination REC; and (v) strains deficient in enzymatic photoreactivation P H R ~4. Recently a number of UV-sensitive strains of N. crassa have been reported (refs. I, I I , 12, 15, 17). Very little is known concerning the biochemical nature of the sensitivity of these strains because of an inability to measure directly the various repair systems operative in Neurospora. Recently we '-'° devised a procedure to specilically label cytosine in the DNA of Neurospora and then measure the excision and photoreactivation of cytosine-containing dimers. We have expanded the utility of this procedure and now report the results of experiments designed to elucidate the biochemical nature of the various UV-sensitive strains of Neurospora with respect to excision repair and photoreactivation. MATERIALS AND METHOI)S

Strains All strains used in this study, were kindly provided by F. J. DE SEre{US. Table I summarizes pertinent information concerning strains used in this study. Growth and culture conditions Conidia from 4- to 6-day slants were inoculated into 2 flasks containing 20 ml of Fries minimal medium supplemented with vitamins (including pantothenate). Tile cultures, containing approximately lO 6 conidia per ml, were incubated with shaking at ambient temperature for 16-i8 h. The DNA was labeled as described by WOI~THY AND EPLER 2°.

Formation of mycdial spheroplasts The radioactive mycelia were harvested over platinum wire filters and the mycelial mat was washed with IO ml of o.o2 M sodium phosphate (pH 6.8). The mycelial mat was then transferred to a 25-ml flask containing 5.o ml of lO% sucrose TABLE I U L T R A V I O L E T - S E N S I T I V E STRAI NS OF Ncl¢rospora £Yassa

Genotype

Strain

Phenotype

74-OR23- i A 74-OR264-25A 74-OR-244-3A 74-OR254-aA 74-OR26o-44 A 74-OR27o-Io4A 74-OR256-2A

w+

wild-type

uvs- r uvs-2 uvs-3 uvs-5 uvs-6 upr-z

pan-2, pan-a, pan-e, pan-e, pan-2, pan-2,

col-2, cot-2, cot-2, cot-2, cot-2, cot-2,

al al al al al al

3lutagen

Isolated by ( R @ )

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U V - s E N S I T I V E MUTANTS OF NEUROSPORA

169

in 0.02 M sodium phosphate (pH 6.0) (LEEF, personal communication), and glusulase (Endo Labs., Garden City, N.Y.) was added to a final concentration of lO%. The suspension was incubated at 3 °° for 2 h with shaking. The spheroplasts were harvested by centrifugation, washed in minimal medium containing lO% sucrose, and resuspended in 4.0 ml of the same medium for irradiation.

Irradiation and postirradiation conditions The conditions for irradiation and the in vivo assays for excision and photoreactivation have been previously described 2°. Assay for biological survival Conidia from 4- to 6-day cultures were harvested and filtered through cotton to remove mycelial fragments. The conidia were washed in sterile water and resuspended in sterile water to a concentration of lO 6 conidia per ml. The suspension was irradiated in a quartz cuvette with a germicidal lamp situated to deliver an incident dose rate of 50 erg/mm2/sec. A correction was made for stirring according to MOROWITZs. Samples were removed at the desired dose, diluted, and plated on Fries minimal medium supplemented as described above plus 1.5% sorbose, o.1% fructose, o.1% glucose, and 1.5% agar. The plates were read after 3 days' incubation at room temperature in the dark. Each strain was checked for the UV-sensitive character before use. RESULTS

Formation of spheroplasts One of the problems inherent on UV studies of fungi is that, due to the mycelial morphology, it is impossible to administer a uniform dose of UV light to a culture. We have found that incubation of an overnight culture of Neurospora with a preparation from the gut of Helix pomatia (glusulase) results in the dispersion of the mycelial m a t into small fragments. These fragments are somewhat osmotically sensitive and will begin to lyse in buffer after 60-90 rain. Viability and respiration studies indicate that treatment of mycelia with glusulase for up to 4 h in a medium with appropriate osmotic stability does not affect viability or respiration (LEEF, personal communication). Fungal strains of typical morphology can be used to assay for excision repair and photoreactivation in the same manner as was reported for the morphological variant, slime m u t a n t 2°. Therefore with certain modifications we are able to extend the utility of our initial observations to include any strain of Neurospora or other fungi. Excision repair in UV-sensitive strains of N. crassa The available UV-sensitive strains were assayed for their ability to remove cytosine-containing dimers from acid-precipitable material in the dark after exposure to a UV dose of 500 erg/mm ~(Fig. I). This dose formed approximately the same number of dimers (between O.lO% and o.12% of total label) in the DNA's of all strains tested and is near the minimum dose required for detection of dimers. It is apparent from Fig. I that three classes of response are represented in the UV-sensitive strains: excision-proficient strains are uvs-r, uvs-5, and uvs-6; excisiondeficient strains are uvs-2 and upr-~; and uvs-3, which shows a slight amount of

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T . E . WORTHY, J. I,. I';PLER

excision, f o r m s t h e t h i r d class. C o m p a r i s o n of t h e slopes of t h e e x c i s i o n - p r o f i c i e n t s t r a i n s to w i l d - t y p e i n d i c a t e s t h a t o n l y u v s - z s h o w s a d e c r e a s e d r a t e of excision. T h e d e c r e a s e d r a t e a p p e a r s to be real, b u t it is p r o b a b l y of little significance since t h e s u r v i v a l of u v s - z is a l m o s t t h e s a m e as w i l d - t y p e . T h e s l i g h t l y r e d u c e d s u r v i v a l c o u l d c o n c e i v a b l y reflect t h e slower r a t e of excision. T h e r e d u c e d r a t e of excision seen in u v s - 3 is s i m i l a r to t h a t f o u n d in U V s e n s i t i v e s t r a i n s of b a c t e r i a such as E. coli B~ a (ref. i3) w h i c h also e x h i b i t s a low TABLE 1[ SUMMARY

OF REPAIR

CAPAHILITIES

Strain

PhenotyDc

74-OR23-1A 744)R264-25 A 74 OR244-3 A 74-OR254-2A 74-OR26o-44A 74-OR27o-Io4 A 74-OR256-2A

uvs-z uvs-2 uvs-3 uvs 5 uvs-6 upr-1

w

( ) I ~" U L T R A V I O L E T - S E N S I T I V E

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In vivo repair system Excision Photoreactiration

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Fig. i. In vivo assay of the excision of pyrimidine dimers in wild-type and U\'-sensitive strains of N. crassa. Conidia were grown for i 6 - i 8 h in liquid minimal medium. Mycelial spheroplasts were made, UV-irradiated (5oo erg/Inm=), and incubated in minimal medium containing ioq,, sucrose in the dark at 3o% The DNA was analyzed for the amount of dimers relnoved after increasing periods of incubation in the dark. The atnount of dimers liresent at zero time ~as aI~proxinIately, O . 1 (~).

l e v e l of excision. T h e low level seen in u v s - 3 m a y be a r e f l e c t i o n of e i t h e r a d e c r e a s e d a f f i n i t y of an e n z y m e s i m i l a r to t h e " U V - e n d o n u c l e a s e " for t h e p y r i m i d i n e d i m e r s u b s t r a t e or a r e d u c e d l e v e l of t h e e n z y m e s a s s o c i a t e d w i t h e x c i s i o n repair. P h o t o r e a c t i v a t i o n in U V - s e n s i t i v e s t r a i n s o f N . crassa

T h e results of an e x p e r i m e n t d e s i g n e d to a s s a y i n vivo for t h e a b i l i t y of U V s e n s i t i v e s t r a i n s of N . c r a s s a to m o n o m e r i z e d i m e r s in t h e p r e s e n c e of l o n g u l t r a v i o l e t r a d i a t i o n f r o m a b l a c k light is seen in Fig. 2. All of t h e s t r a i n s p h o t o r e a c t i v a t e t h e i r

U V - s E N S I T I V E MUTANTS OF NEUROSPORA

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Fig. 2. I n vivo a s s a y for p h o t o r e a c t i v a t i o n of p y r i m i d i n e d i m e r s in w i l d - t y p e a n d U V - s e n s i t i v e s t r a i n s of N . crassa. Conidia w e r e g r o w n for 16-18 h in liquid m i n i m a l m e d i u m . Mycelial s p h e r o p l a s t s w e r e m a d e , U V - i r r a d i a t e d (I oo e r g / m m ~ ) , a n d e x p o s e d to blacklight. T h e D N A w a s a n a l y z e d for t h e a m o u n t of d i m e r s r e m a i n i n g . T h e a m o u n t of d i m e r s p r e s e n t a t zero t i m e w a s a p p r o x i m a t e l y O.2~o.

DNA to approximately the same extent. From the survival data, upr-z had been thought to be defcient in photoreactivationlL but in this experiment we found that this strain could photoreactivate dimers in vivo. A recent report by TUVESONTM also indicates that in vitro enzyme activity is present. The possibility exists, however, that the deficient or impaired photoreactivation initially seen in upr-z is a manifestation of conditions present in the conidia. For example, there m a y be little or no enzyme present in conidia of upr-z, or perhaps the enzyme is inaccessible to the DNA due to a structural modification in the conidia. This alternative should be susceptible to testing since the enzyme would probably be active in an in vitro assay with a heterologous DNA. DISCUSSION

In this paper we have presented evidence indicating that certain strains of Neurospora fail to repair radiation damage in the form of pyrimidine dimers; in addition, the experiments indicate the existence of strains which can repair pyrimidine dimers as measured by excision and photoreactivation but are, nevertheless, UVsensitive. To aid in understanding these results we will attempt to draw analogies, when applicable, to the well-characterized bacterial repair systems. Although there is a certain danger in extrapolation from one system to another, especially from prokaryotes to eukaryotes, we feel that the insight gained from such an extrapolation greatly outweighs the risk involved. At the outset, it is imperative to emphasize that any analogies must, at this time, be tentative; confirmation must await further studies. Table I I summarizes the postirradiation repair and survival capabilities of the available UV-sensitive mutants of Neurospora. Based on the available data, the six mutants can be classified into three categories analogous to their bacterial counterparts. (±) Three mutants, uvs-2, uvs-3, and upr-z, are classed as uvr because of their observed deficiency in excision repair and their intermediate sensitivity to ionizing

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T . E . WORTHY, J. L. F.I'LliR

r a d i a t i o n (DE NERRES, pers,.mal c o m m u n i c a t i o n ) ; two of these m u t a n t s , uvs-e and upr-z, e x h i b i t no d e m o n s t r a b l e excision, while uvs-3 shows a residual a m o u n t of excision. Uvs-2 is p r o b a b l y analogous to E. coil" Bs a which, although uw', shows a slight a m o u n t of excision ~a. In the light of recent e x p e r i m e n t s i n d i c a t i n g t h a t p r e p a r a t i o n s of UV-endonuclease recognize an u n k n o w n lesion induced b y ionizing r a d i a t i o n under anoxic conditions", the o b s e r v e d s e n s i t i v i t y to ionizing r a d i a t i o n l n a y be due to the i n a b i l i t y of such strains to excise the " g a m m a - r a y lesion" and not to an i n a b i l i t y to repair s t r a n d - b r e a k s . The i n t e r m e d i a t e s e n s i t i v i t y of these strains to ionizing radiation s u b s t a n t i a t e s this hypothesis since a true sensitivity, based on an i n a b i l i t y to r e p a i r d a m a g e i n d u c e d b y such r a d i a t i o n , should confer g r e a t e r s e n s i t i v i t y t h a n is observed. F u r t h e r , genetic analysis of these m u t a n t s indicated t h a t t h e y tire single m u t a t i o n s n,*~,~. (e) Two m u t a n t s of Neurospora, m,s-I a n d uvs-5, are c a p a b l e of both excision a n d p h o t o r e a c t i v a t i o n a n d are the least sensitive to UV of the available m u t a n t s . Unless t h e y are deficient in an u n k n o w n repair system, we feel t h a t t h e y are p r o b a b l y similar to the Ion m u t a n t s of E. coil, which are i n c a p a b l e of c o m p l e t i n g cell division after exposure to UV 7 a l t h o u g h the uvs- 5 m u t a n t is not sensitive to ionizing r a d i a t i o n (DE SERRES, personal coinmunication). I t is conceivable t h a t in the two m u t a n t s one or more steps in conidial g e r m i n a t i o n are sensitive to U\:. (3) The t h i r d class of n m t a n t s sensitive to UV is uvs-6, which is proficient in b o t h excision a n d p h o t o r e a c t i v a t i o n . It is, however, the most sensitive to ionizing r a d i a t i o n (DE SERRES, personal communication). The basis of the observed UVs e n s i t i v i t y is not known, b u t we propose t h a t it m a y be a consequence of a rec m u t a tion. E x p e r i m e n t s are u n d e r w a y in our l a b o r a t o r y to e x a m i n e the effect of this gene on r e c o m b i n a t i o n . W i t h the t e n t a t i v e classification of the UV-sensitive m u t a n t s of N . cyassa, it is of interest to e x a m i n e the consequences of such s e n s i t i v i t y in t e r m s of their effects on such p h e n o m e n a as genetic r e c o m b i n a t i o n a n d m u t a t i o n induction. U n f o r t u n a t e l y m o s t of the m u t a n t s are either sterile in h o m o z y g o u s crosses (nvs-2 and uvs-5) or show no effect on recomt)ination (uvs-z, uvs-2, a n d uvs-4)1,10,15. M u t a t i o n i n d u c t i o n studies b y DF. SJ~:RRES~ a n d DE SERRES A~
U V - S E N S I T I V E MUTANTS OF NEUROSPORA

173

concerning the role of repair in mutation induction and genetic recombination can be considered. ACKNOWLEDGMENTS

This investigation was sponsored by the U.S. Atomic Energy Commission under contract with the Union Carbide Corporation. We thank J. L. LEEF, ALICE L. SCHROEDER, and F. J. DE SERRES, for advice and discussion; and R. F. KIMBALLand J. F. LEMONTTfor their critical review of the manuscript. One of us (T.E.W.) was a Laboratory Graduate Participant from the University of Tennessee under appointment from the Oak Ridge Associated Universities. This work is in partial fulfillment of the requirements for the Ph.D. degree at the University of Tennessee, Knoxville, Tennessee. REFERENCES I CHANG, L. T., AND R. W. TUVESON, U l t r a v i o l e t - s e n s i t i v e m u t a n t s in Neurospora crassa, Genetics, 56 (1967) 8Ol-8IO. 2 DE SERRES, F. J., M u t a b i l i t y of U V - s e n s i t i v e s t r a i n s of Neurospora crassa, Genetics, 68 (197 I) sI 4. 3 DE SERRES, F. J., AND M. E. SCHUPBACH, T h e effect of U V - s e n s i t i v e m u t a n t s of Neurospora crassa on r a d i a t i o n - i n d u c e d i n a c t i v a t i o n a n d m u t a t i o n i n d u c t i o n at specific loci, Abstracts of the 2nd Annual Meeting of the European Environmental Mutagen Society, ~ I N K O V Y Castle, I972. 4 HAYNES, R. H., T h e i n t e r p r e t a t i o n of microbial i n a c t i v a t i o n a n d r e c o v e r y p h e n o m e n a , Radiation Res., Suppl., 6 (I966) i 29. 5 HAYNES, R. H., R. M. BAKER AND G. E. JONES, Genetic i m p l i c a t i o n s of D N A repair, in G. O. PHILLIPS (Ed.), Energeties and Mechanisms in Radiation Biology, A c a d e m i c Press, N e w York, I968, pp. 425-465 ` 6 HOWARD-FLANDERS, P., AND R. P. BOYCE, D N A repair a n d genetic r e c o m b i n a t i o n : S t u d i e s on m u t a n t s of Eseherichia coli defective in t h e s e processes, Radiation Res., Suppl., 6 (I966) I56--I 85 . 7 HOWARD-FLANDERS, P., R. P. BOYCE AND L. THERIOT, T h r e e loci in Escherichia coli K-I2 t h a t control t h e excision of p y r i m i d i n e d i m e r s a n d c e r t a i n o t h e r m u t a g e n p r o d u c t s f r o m D N A , Genetics, 53 (I966) I I I 9 - I I 3 6 . 8 MOROWITZ, H. J., A b s o r p t i o n effects in v o l u m e irradiation of m i c r o o r g a n i s m s , Science, i I i (195 o) 229-23o. 9 PATERSON, M. C., AND R. B. SETLOW, A n e n d o n u c l e o l y t i c a c t i v i t y f r o m Micrococcus luteus t h a t acts on T - r a y - i n d u c e d d a m a g e in p l a s m i c D N A of Escherichia coli minicells, Proc. Natl. Acad. Sci. (U.S.), 69 (I972) 2927-293I. io SCHROEDER, A. L., U l t r a v i o l e t - s e n s i t i v e m u t a n t s of Neurospora I. Genetic basis a n d effect on r e c o m b i n a t i o n , Mol. Gen. Genet., io 7 (i97oa) 2 9 i - 3 o 4. i i SCHROEDER, A. L., U l t r a v i o l e t - s e n s i t i v e m u t a n t s of Neurospora II. R a d i a t i o n studies, Mol. Gen. Genet., io 7 (197ob) 3o5-32o. i2 SCHROEDER, A. L., F. J. DE SERRES ANn M. E. SCHOPBACH, A n e w uvs m u t a n t , Neurospora Newsletter, 19 (I972) 17. 13 SETLOW, R. B., R e p a i r of D N A , in V. V. KONINGSSERaER AND L. BOSH (Eds.), Regulation of Nucleic Acid and Protein Biosynthesis, Elsevier, A m s t e r d a m , 1967, pp. 51-62. 14 SMITH, K. C., T h e role of genetic r e c o m b i n a t i o n a n d D N A p o l y m e r a s e in t h e repair of d a m a g e d D N A , in A. GIESE (Ed.), Photophysiology, Vol. 6, A c a d e m i c Press, N e w York, 1971, pp. 209278. 15 STADLER, D. R., AND D. A. SMITH, A n e w m u t a t i o n in N e u r o s p o r a for s e n s i t i v i t y to ultraviolet, Can. J. Genet. Cytol., io (I968) 916-919. 16 TUVESON, R. W., Genetic a n d e n z y m a t i c a n a l y s i s of a gene controlling U V s e n s i t i v i t y in Neurospora crassa, Mutation Res., 15 (1972) 411-424. 17 TUVESON, R. W., AND J. MANGAN, A U V - s e n s i t i v e m u t a n t of N e u r o s p o r a defective for p h o t o r e a c t i v a t i o n , Mutation Res., 9 (I97 o) 455-466.

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18 UNRAU, P., R. WHEATCROFT AND B. S. Cox, M e t h o d s for tile a s s a y of u l t r a v i o l e t l i g h t - i n d u c c d p y r i m i d i n e d i m e r s in Saccharomyces cerevisiae, Biochim. Biophys. Acta, 269 (I972) 3I I 32I. 19 ~VITKIN, E. M,, U l t r a v i o l e t - i n d u c e d m u t a t i o n and I)N A repair, 3~2n. R,'~,. (A,m't.. 3 (,o~)()) 525 552 • 20 V~rORTHY,T. E., AND J. L. I:PLER, R e p a i r of u l t r a v i o l e t l i g h t - i n d u c e d d a m a g e to t he dcoxyrib(~ nucleic acid of Neurospora crassa, .]. Bactcriol., i i o (i972) i o l o i o i 0 .