Mutation Research, 29 (1975) 149-153 <~) Elsevier Scientific Publishing Company, Amsterdam--Printed in The Netherlands 149

Further evidence for an inducible recombination repair system in Ustilago maydis

Introduction The frequency of recombination in mitotic diploids of fungi is two to three

orders of magnitude lower than that observed during meiosis 8. I t has therefore been suggested that one or more of the enzymes or proteins required for recombination are induced during meiosis and repressed during mitotic division 3. However, the frequency of mitotic recombination is greatly increased after irradiation, particularly gene conversion in heteroallelic diploids of U. m~vdis or Sacch~romyces cerevisiael, 12. I t is believed that this is due to a recombination repair mechanism, since certain radiation sensitive mutants show little or no increase in gene conversion after irradiation2,11. If an enzyme essential for recombination repair is repressed in mitotic cells, then irradiation could trigger its synthesis.

More information was gained about allelic recombination a f t e r / - r a y treatment by using the nitrate reductase system in U. maydis ~. I t is possible to measure re- combination in diploids heteroallelic for the structural gene for the enzyme by de- tecting very low levels of active enzyme synthesized from the recombinant gene. In normal procedures, recombination is only detected in viable cells, since the phenotype is scored by allowing colonies to grow, but this biochemical method allows recombi- nation to be detected soon after it has occurred, in either viable or non-viable cells. I t was found that ionising radiation increased the amount of recombination in the whole population up to a dose of 450 krad, but thereafter it declined. The frequency of recombination measured by nitrate reductase activity closely paralleled the frequency determined by recombinant colony counts per total cells plated, but was quite different from the calculated frequency after correcting for inviability. In other words, after high doses of radiation most of the recombination occurred only in viable cells. This established an unexpected relationship between recombination and the ability to survive.

Earlier experiments had shown that increasing doses of UV-irradiation pro- gressively inactivated the inducible structural gene for nitrate reduetase in U. maydis lo. The doses necessary for inactivation of this single gene were in fact not dissimilar to those which killed the cells. This observation suggests an explanation for the recombination experiments. If recombination repair is an inducible process, then increasing doses of radiation will inactivate the repressed structural gene(s) before they can be transcribed. A cell in which this happens will then have no recombination repair mechanism and is likely to be non-viable. A cell in which the structural gene remains intact, or has been effectively repaired by an excision repair pathway, has a much greater chance of survival*.

Additional evidence for an inducible repair mechanism in U. maydis comes from the discovery of UV recovery in certain radiation sensitive pyrimidine auxotrophs which is reported by MOORE v. Low doses of UV progressively kill cells of pyr I - I , but recovery is seen as the dose is increased further.

If an inducible mechanism exists, it should be possible to block it with cyclo-

Abbreviations: CH, cycloheximide; CM, complete medium; NM, nitrate minimal medium; PR, photoreactivation.

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Fig. i. The effect of holding cells in CH for increasing lengths of t ime after UV irradiation. Cells of diploid d58 were grown in complete medium (CM), harvested in log phase, washed and t reated with 9o J" n1-2 UV. Cells were suspended in water containing 5/ ,g/ inl CH, and samples were removed at intervals to measure survival (O) on CM or allelic recombinat ion (0) on supplemented ni t ra te minimal medium (NM). d58 is heteroallelic at the n a t i , n i c i and i n o s i loci, bu t in this experiment only n a t + recombinants were scored. The dose of UV used increases the recombinat ion frequency approx. 4o-fold. For a full description of media and methods employed, see ref. 5.

Fig. 2. Survival of cells held in water or CH for 9 h after irradiation. Strain diploid d66 was grown ill CM, harvested in log phase, washed and irradiated. Cells were suspended in water with or with- out 5 / , g / m l CH and plated on CM.

heximide (CH), a po ten t inhibi tor of prote in synthesis in eukaryo t i c cells. I describe here exper iments which confirm this predict ion. The procedures used are given in the legends to the figures, or descr ibed elsewhere'%

Results The first essential is to de te rmine the op t imum length of t ime to hold cells in CH

after UV. Clearly if the t ime is too short, p ro te in synthesis can s imply resume af ter the cells are p laced in normal medium, and no effect will be seen. The exper iment in Fig. I shows tha t cells removed from the block in prote in synthesis up to 5 h af ter i r rad ia t ion reta in most of thei r viabi l i ty , bu t thereaf te r t hey are progress ively killed. The kinetics of survival are essential ly the same for the whole cell popula t ion and for UV induced recombinants . In subsequent exper iments the i r r ad ia t ed cells were held in CH for a t least 8 tl before rescue. I t is also necessary to check ti le effect of CH on un t r ea t ed cells: in several exper iments log phase popula t ions were 8o% viable af ter 9 h CH t rea tment .

The d rama t i c effect of CH holding on surv iva l af ter UV is shown in Vig. 2. In this exper iment control cells were held in water , but since there is no significant l iquid holding recovery in wi ld - type cells of U. maydis, the same survival would have been seen if the cells had been p la ted d i rec t ly af ter UV. To test the effect of CH holding on recombinat ion , heteroallel ic cells were i r r ad ia ted and then t ransfer red to CH at in tervals thereaf te r and held for 8 h before rescue. I t can be seen from Fig. 3 tha t CH blocks at least 8o% of the UV s t imula ted recombinat ion , p rov ided it is added within

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TIME (HP, SJ OFADDITION OF CH AFTER UV T i M E (HRS.) AFTER UV B E F O R E PR

Fig. 3. Effect of CH t r ea tmen t on UV induced allelic recombination. Cells of diploid d58 were grown in CM, harvested in log phase, washed and irradiated wi th 90 J ' m s UV. Cells were held in water until t ransferred to 5/~g/ml CH. After 8 h cells were plated on supplemented NM to select for recombinants and on CM to measure viability. Frequencies of recombinat ion are per cells plated ( - - e - - ) and per viable cells (---©---) .

Fig. 4. The effect on recombinat ion of photoreact ivat ion at increasing t imes after UV t rea tment . Cells of diploid d58 were grown in CM to s ta t ionary phase, washed and irradiated with an incident dose of 7oo J . m -2 UV. Cells were spread on supplemented NM and treated at the t imes indicated for 3 ° min with two black light fluorescent lamps. Survival in this exper iment was lOO% for P R immediately after UV, declining steadily to 6o% for P R at 9 h after UV. m m r

1.5--2 hours after irradiation. Later than this it has progressively less effect. From the experiments with nitrate reductase already mentio ned, it is known that 7-ray induced recombination is completed by 4-5-5 hours after irradiation. (For technical reasons, comparable experiments cannot easily be carried out with UV). With regard to sur- vival, experiments have also been done which show that the effect seen in Fig. 2 occurs only if CH is added within 1.5-2 hours after UV irradiation.

Photoreactivation (PR) makes it possible to determine when recombinagenic pyrimidine dimers in DNA are removed. In the experiments shown in Fig. 4, cells were treated with a low dose of UV and photoreactivated at intervals thereafter. About 6o% of the recombination is abolished by immediate PR (either because all the dimers are not split, or because there are recombinagenic lesions other than pyrimidine dimers), but the effect steadily decreases after about 2 h showing that the dimers are being removed by a recombination process.

D i s c u s s i o n

These results, together with those previously published, show that most allelic recombination occurs sometime between 2-5 h after irradiation, and that protein synthesis is required within about 2 h after UV treatment for full survival and production of recombinants. Studies with radiation sensitive mutants of U. m a y d i s

indicate that there are at least two dark repair pathways.The mutant uvs 3 is defective in the rapid excision of pyrimidine dimers, but is capable of removing them after a lag of about 2 h la. This mutant is known to be proficient in recombination 2. Other mutants, rec I and rec 2, rapidly excise dimers from DNA la, but are defective in recombinationS, 6. UNRAU la has suggested that the excision and recombination repair mechanisms act sequentially. If it is essential for them to do so to achieve maximum

152 SHORT COMMUNICATIONS

repair efficiency, then split dose experiments will not necessarily provide any evidence for or against an inducible repair mechanism.

RADMAN 9 a n d WITKIN AND GEORGE 14 have presented strong argunients for an inducible "SOS" repair nmchanisin in Escherichia coli, components of which are required for UV reactivation, UV mutagenesis and prophage induction. If inducible repair mechanisms do exist, a crucial question is the lnechanism of inactivation of repressor molecules after irradiation. For equivalent doses of UV, excisionless mutants of E. coli have a higher level of SOS repair than wild-type cells'%'L and the same is true for the excisionless nlutant, uvs 3, and wild-type U. mavdis with regard to UV stinmlated recombination 2. This suggests that it is the presence of pyrimidine dimers in DNA wtlich is responsible for repressor inactivation. The same conclusion has been reached from the study of UV recovery in a pyrimidine auxotroph of U. mavdisL One simple possibility is that repressor molecules have a greater affinity for DNA con- taining damaged bases than for their operator binding sites.

Diploids homozygous for the mutant rec I in U. mavdis (previously designated uvs i) have a higher than normal spontaneous frequency of mitotic recombination, and very little or no induced recombination after irradiation 'a. It is therefore tempting to suppose that this gene codes for a repressor which does not bind as tightly as usual to its operator regions, thereby allowing a degree of "constitutive" mitotic recombi- nation, and it does not bind at all to damaged DNA, thereby preventing induced recombination. The possibility will be explored more fully in a paper 6 which describes in full the complex phenotype of rec • strains, and PAUL [7NRA uand PETER MOORE

for encouraging me to publish these results. I t h a n k MAR~ WYNN EVANS for her t e c h n i c a l a s s i s t a n c e .

2\rational Institute for Medical Research Mill Hill, London NFV 7 ±AA (Great Britai,z)

ROBIN HOLIADAY

I HOLLIDAY, R., Studies oi1 mitotic gene conversion in ([stilago, Genet. lees., 8 (I966) 323 337. 2 HOLLI1)AY, R., Altered reconlbination frequencies in radiation sensitive straius of Ustilago,

M u t a t i o n R e s . , 4 (19671 275 288. 3 HOLLIDAY, R., Geuetic reconlbination in fungi, in W. J. PEACOCK AND R. l). P, ROCK (V.ds.),

Replication and Recombinatio~ o] Genetic J~Iaterial, Australian Academy of Science, Canberra, I968, pp. 157 174.

4 HOLLIDAY, R., Biochenlical measure of the tilne and frequency of radiation-induced allelic recombination in Ustilago, Nature (London) , 232 (~97 I) 233-236.

5 HOLLIDAY, l{., Ustilago maydis, in R. C. KING (Ed.), Handbook of Genetics, Plenum, New York, 1974, pp. 575 595.

6 HOLLIDAY, R., R. 1'.'. HALLIWELL, V. ROWELL AND M. \V. EVAyS, Abnormal reconlbination in yec I strains of U. maydis, in preparation.

7 MOORB, P. D., Radiation sensitive pyrimidine auxotrophs of Ustilago maydis, l l. A s tudy of repair ulechanisms and UV recovery in pyr i, Mutat ion Res., 28 (I9751 367 38o.

8 POYTECORVO, G., Trends in Genetic Analys is , Oxford University Press, London, 1958. 9 I{ADMAN, M., Phenomenology of an inducible mutagenic D N A repair pa thwa y in Escherichia

coli: SOS repair hypothesis, in M. MILLER (Ed.), Molecular and Environmental d specls of M u - tagenesis, Thomas, Springfield, Illinois (in press).

Io RESNICK, M. A., AND IR. HOLLIDAY, Genetic repair and the synthesis of nitrate reductasc in Ustilago maydis after t i V irradiation, 3ioi. Gen. Genet., i 1 I (I9711 I 71 I 8 4 .

11 RODARTE RAMON, U. S., AND R. N. MORTIMER, Radiation induced recombination in Saccharo- nlyces: isolation and genetic study of recombination deficient mutants , Radial. tees., 49 (I97 I) 133 147.

12 P, OMAN, H. L., AND 1;. JACOB, A comparison of spontaneous and ultraviolet induced allelic recombination with reference to the reconlbination of outside markers, Cold Spring Harbor £'ymp. Quant. Biol., 23 (1958) I55--I60.

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13 UNRAU, P,, The excision of pyrimidine dimers from the DNA of mu tan t and wild-type strains of Ustilago, Mutation Res., 29 (1975) 53-65.

14 WITKIN, E. M., AND D. L. GEORGE, UV mutagenesis in po l lA and uvrA polA derivatives of E. coli B/r: evidence for an inducible error prone repair system, Genetics, Suppl. 73 (1973) 91-1o8.

R e c e i v e d F e b r u a r y I 8 t h , 1975