Molee. gen. Genet. 142, 209--224 (1975) © by Springer-Verlag 1975
DNA Polymerase of Ustilago maydis: Partial Characterization of the Enzyme and a pol 1 Mutation Penelope A. Jeggo*, and Geoffrey 1~. Banks National Institute for Medical Research, Mill Hill, London EW7 tAA, England Received August 5, 1975
Summary. The major DNA polymerase activity of wild-type U. maydis has been extensively purified. It possesses a molecular weight of about 150,000 daltons and appears to require a DNA primer with a 3'-hydroxyl terminus as well as a template. The polymerase activity has also been purified from the pol 1-1 strain, which is temperature sensitive for growth and DNA synthesis, and which at the restrictive temperature contains only 10-25 % levels of the DNA polymerase activity obtained from wild-type strains. It was similar in all properties studied, except that the activity was thermolabile at 40° C compared to that from the wildtype strain. Physiological studies on the mutant showed that it was only slightly sensitive to UV, ionising radiation and nitrosoguanidine at the permissive temperature, and was proficient in genetic recombination. The results suggest that the pol 1-1 gene product does not play an important role in repair and recombination processes within the cell, and that its primary function lies in replication. Introduction DNA polymerases have now been isolated and characterised from a wide range of organisms and their tissues. Three well studied bacteria, Escherichia coli, Bacillus subtilis and Micrococcus luteus, each possess three DNA polymerases (Kornberg and Gefter, 1970, 1971, 1972; Wickuer et al., 1972; Gefter et al., 1972; Otto etal., 1973; Ganesan et al., 1973; Bazill and Gross, 1973; Hamilton, 1974), and some advances have been made towards an understanding of their role in the cell (Gefter et al., 1971; Nusslein et al., 1971; Keumpel and Yeomett, 1970; Okazaki et al., 1971 ; Gass et al., 1973 ; Hamilton et al., 1974 ; Brutlag and Kornberg, 1972). Two nuclear and a separate mitochondrial enzyme have been reported from yeast (ttelfman, 1973; Wintersberger and Wintersberger, 1970a, b), but little is known of their cellular functions. Multiple molecular weight DNA polymerases have been obtained in different intracellular locations in several mammalian systems (Chang and Bollum, 1971 ; Baril et al., 1971 ; Weissbach et al., 1971) and an attempt has been made to study their functions by estimating the variations in their levels in tissues actively involved in DNA synthesis (Chang and Bollum, 1972; Chang et al., 1973). I n general, the correlation between enzyme functions and biological processes in eukaryotic systems has depended on physiological means, since genetic techniques analogous to those using the bacterial DNA polymerase mutants have not been available.
* Present Address: Imperial Cancer Researeh Fund, Burtonhole Lane, Mill Hill, LondonNW7 1AA, England.
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P.A. Jeggo and G. R. Banks
The basidiomycete fungus, U. maydis is a yeast-like e u k a r y o t e which has been used extensively i n genetic a n d biochemical studies (Holliday, 1974; H o l l i d a y et al., 1974). The charaeterisation of several m u t a n t strains of this organism which are t e m p e r a t u r e sensitive for DlkTA synthesis has b e e n described ( U n r a u a n d Holliday, 1970). I n particular, it was shown t h a t crude extracts from t h e m u t a n t pol 1-1 c o n t a i n reduced (10-25 %) levels of D N A polymerase a c t i v i t y c o m p a r e d to those from t h e wild-type s t r a i n after i n c u b a t i o n of t h e cells a t t h e restrictive t e m p e r a t u r e . F u r t h e r m o r e , after growth at t h e permissive t e m p e r a t u r e , t h e part i a l l y purified D N A polymerase a c t i v i t y was f o u n d to be heat labile (Jeggo et al., 1973). T h e evidence suggests t h a t t h e pol 1-1 m u t a n t m a y be t e m p e r a t u r e sensitive for D N A synthesis in vivo because it possesses a t e m p e r a t u r e sensitive D N A polymerase, which is t h e replicative e n z y m e for this organism. I n this p a p e r we describe some physiological properties of this m u t a n t , a n d r e p o r t on a f u r t h e r purification a n d t h e i n i t i a l charaeterisation of t h e enzymes from t h e wild-type a n d pol 1-1 strains. A fuller a c c o u n t is g i v e n b y Jeggo (1973).
Materials a n d Methods
1. Strains Strains were obtained from stock cultures (see Holliday, 1974). Strains used were wildtype a2bl; rec 1-1 a2bl; rec 2 a~bl; uvs 3 a2bz. Various temperature sensitive strains were provided by P. Unrau. ala e and bib2 are mating type alleles, rec denotes deficiency in recombination, and uv8 sensitivity to UV light.
2. Media Cells were routinely grown in either nitrate minimal medium or complete medium as described by Itolliday (1961 a, b, 1974). For solid medium, New Zealand Davis agar was added at 20 g per litre. Mating medium consisted of 1.7 g 'Difco' cornmeal agar, 1 g Davis agar and 1 g activated charcoal added to 100 ml CM.
3. Cetl-t~ree Extracts Cells were grown in liquid complete medium to late log phase (1 × 107 cells/ml) after inoculation from a starter culture. Cell counts were made with a Coulter counter Model A after appropriate dilution. Cultures (routinely 1 litre) were harvested and washed twice in extracting buffer (0.01 phosphate pit 8.0, 0.001 M EDTA, 0.001 M MSH) at 4° C in a refrigerated centrifuge (Sorvall RC-2B) at 9,000 rpm for 5 rains. Washed cells were resuspended in 3 ml extracting buffer, half a volume of cold acid-washed Ballotini beads (no. 11) added, and the cells disintegrated by shaking in a Mikkle disintegrator for 10 rains. Beads and cellular debris were removed by ccntrifugation at 9,000 rpm for 20 rains, after which the supernatant was centrifuged 37,000 rpm for 1 hr in a Type 65 head in a Beckman L2-65B ultracentrifuge. All stages were carried out at 2-4 ° C. The supernatant from the final centrifugation was used as a crude cell-free extract.
4. Large Scale Culture Large quantities of wild-type haploid cells, strain a~bl, were grown in 300 L medium containing 3 kg Oxoid Yeast Extract, 6 kg Oxoid Bacteriological Peptone, 6 kg sucrose and 50 ml Silicone R.D. at 32° C. The cells were harvested in late log phase by centrifugation in a Sharples Super centrifuge Model 6A at 15,000 rpm, and stored as a pellet at --20 ° C. Large quantities of the mutant strain (pol 1-1) were grown overnight at 22°C to 1 × 10~ cells/ml, par~ harvested and the remainder incubated for 5 hrs at 32° C.
DNA Polymerase Deficient Mutant of Ustilago maydis
211
5. Enzymes and Substrates Pancreatic DNase (DNase 1), micrococcal DNase and calf thymus DNA were purchased from the Worthington Biochemical Corp. Phosphocellulose (P 11) was from the Whatman Co. deoxyribonucleoside triphosphates from PL Biochemieals Inc. and [8tt]-thymidine and [aH]d T T P (11.2 Ci/mM) from the Radiochemical Centre.
6. D N A Polymerase Assay The reaction measures the incorporation of [aH]-dTTP into acid insoluble DNA. The mixture contained 10 ~mole Tris-HC1 p H 7.5, 5 nmole each of dATP, dCTP and dGTP, 0.5 nmole dTTP, 0.1 nmole [3H]-dTTP (1.25 ~zCi), 20 ~zmole KCI, 1.5 fxmole MgCl~, 75 ~xg heat denatured calf thymus DNA and aliquots of the enzyme in a total volume of 0.15 ml. After incubation at 32 ° C for 40 rain, 0.1 ml of the mixture was pipetted onto a Whatman 3 mm filter paper square, which was immediately immersed in cold 5 % TCA containing 1% sodium pyrophosphato. The filters were washed in two further changes of TCA and finally in cold 95 % ethanol, dried and counted in scintillation fluid containing 5 g 2,5-diphenyloxazole (PPO) and 0.2 g 1,4-bis (2-4-methyl-5-phenyloxazolyl) benzene (dimethyl POPOP) per litre of toluene. One unit of activity is defined as the amount of protein which incorporates 1 pmole of dTTP into acid insoluble DNA under the above conditions.
7. Deoxyribonuclease Assay The method measures the release of acid soluble radioactivity from E. coli [aH]-DNA. The mixture contained 55 fzmole Tris-tIC1 pit 7.2, 0.15 ~mole MgC12, 5 ~g E. coli [3It]-DNA and enzyme in a volume of 0.15 ml. After incubation at 32 ° C for 40 rains, 0.1 ml (1.5 mg/ml) carrier DNA and 0.2 ml cold 5% TCA were added, and after holding in ice for 10 rains, the tubes were centrifuged at 10,000 rpm for 10 rains. 0.1 ml of the supernatant solution was counted in scintillation fluid containing 60 g naphthalene, 4 g PPO, 0.2 g dimethyl P O P O P 100 ml methanol and 900 ml 1.4 dioxan.
8. DIYA Calf thymus DNA was dissolved at 3 mg/ml in 0.2 M Tris-IiC1 p i t 8.0-0.02 M NaC1. I t was denatured by incubation in a boiling water bath for 10 rains, followed by rapid cooling in ice. DNase I nicked calf thymus DNA was prepared as described by Aposhian and Kornberg (1962), and mierococcal nuclease nicked DNA by the method of Richardson and Kornberg (1964). [aHJ-thymidine labelled E. coli DNA was extracted by iVIarmur's method (1961).
9. Other Biochemical Methods Single-stranded calf thymus DNA cellulose was prepared as described by Alberts and Herrick (1971) using Munktell 410 cellulose. About 1.5 mg DNA/g cellulose remained bound after extensive washing. Protein concentrations were determined by the method of Lowry et al. (1951) using bovine serum albumin as the standard.
10. Growth Rates Cells were grown in liquid CM with vigorous aeration. Cell counts were made with a Coulter counter.
11. Irradiation UV irradiation was from a Hanovia low-pressure germicidal lamp delivering 46 ergs per mm 2 per second at a distance of 15 cm. Cells were treated after spreading on CM, or in suspension in sterile water, at l0 s cells/ml. Precautions were taken to prevent photoreactivation.
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P . A . Jeggo and G. R. Banks
F-irradiation was from a Gammabeam 650 Co60 source delivering 40 Krads per rain to the cell suspension. Cells were irradiated at 107/ml in a beaker with stirring.
12. Treatment with N-Methyl N Nitrosoguanidine (NG) Log phase cells were grown in CM at 22 ° C and NG added to a final concentration of 200 ~g/ml. The culture was incubated with shaking at 22 ° C and at 6 rain intervals samples were taken and diluted immediately 100 fold to prevent further mutagenic action. Suitable dilutions were plated on CM and the survival estimated after 5-8 days incubation at 22 ° C.
13. Isolation of Diploids Diploids were isolated by mixing strains of complementary mating types on mating medium, each parent having a different nitritional requirement. When white hyphae had appeared, the plate was replica plated to ~ on which heterokaryons grew very slowly, whereas diploids grew vigorously. Single colony isolates were made of each diploid.
ld. Spontaneous Mutation The spontaneous mutation frequency was measured by a fluctuation test using the method of the median developed by Lea and Coulson (1949). For each strain, 10 tubes each conraining 5 ml CM were inoculated with 103 cells/ml from a log phase starter culture. The parallel cultures were grown for 2-3 days at 22 ° C to stationary phase and suitable dilutions plated as required.
Results
1. Puri/ication o] DNA Polymerase A l l o p e r a t i o n s were c a r r i e d o u t a t 4 ° C a n d c e n t r i f u g a t i o n was a t 9,000 r p m for 20 rains unless o t h e r w i s e i n d i c a t e d . Step I: Preparation o/Crude Extract. 300 g frozen cells were t h a w e d a n d w a s h e d in Buffer A (10 m M Tris-HC1 p H 8.0, 1 m M 2 - m e r e a p t o e t h a n o l a n d 10% glycerol). A f t e r centrifugation, t h e cells were r e s u s p e n d e d in 300 m l Buffer A a n d 600 g cooled a c i d - w a s h e d B a i l o t i n i No. 11 glass b e a d s a d d e d . T h e ceils were disinteg r a t e d in a cooled W a r i n g B l e n d e r u n t i l a b o u t 100% cell b r e a k a g e was o b s e r v e d u n d e r t h e microscope. T h e t e m p e r a t u r e was m a i n t a i n e d below 15 ° C. A f t e r allowing t h e b e a d s t o settle, t h e s u p e m a t a n t was d e c a n t e d a n d t h e d e b r i s w a s h e d twice w i t h 100 m l of Buffer A. T h e r e s u l t i n g s u p e r n a t a n t s were c o m b i n e d a n d c e n t r i f u g e d t o give F r a c t i o n I (170 ml). Step II: Removal o] Nucleic Acids. Nucleic acids were r e m o v e d b y t h e p h a s e s e p a r a t i o n t e c h n i q u e of A l b e r t s a n d H e r r i c k (1971). Solid NaC1 was a d d e d slowly t o s t i r r e d F r a c t i o n I t o a c o n c e n t r a t i o n of 1.7 M. A 30% p o l y e t h y l e n e glycerol ( C a r b o w a x 6000) s o l u t i o n was a d d e d t o a final c o n c e n t r a t i o n of 10% a n d t h e suspension s t i r r e d for 30 rain in ice. A f t e r c e n t r i f u g a t i o n a t 10,000 r p m for 15 mills, t h e s u p e r n a t a n t solution was d i a l y s e d a g a i n s t Buffer B (10 m M Tris-HC1 p t I 8.0, I m M E D T A , 1 m M 2 - m e r c a p t o e t h a n o l , 5 0 m M N a C 1 a n d 10% glycerol) (51itre w i t h 2 changes). F i n a l l y , t h e e x t r a c t was clarified b y c e n t r i f u g a t i o n a t 37,000 r p m in a T y p e 40 r o t o r on a B e c k m a n L2-65B centrifuge for 60 mins t o give T r a c t i o n I I (300 ml). Step III: DNA-cellulose Chromatography. I t h a d p r e v i o u s l y b e e n shown t h a t D N A p o l y m e r a s e a c t i v i t y f r o m U. maydis e l u t e d f r o m a DNA-eellulose c o l u m n
DNA Polymerase Deficient Mutant of Ustilagomaydis
213
at 0.dM NaC1. To facilitate the large scale preparation of purified D N A polymerase, the enzyme was eluted from the DNA-cellulose column in a stepwise manner. Fraction I I was applied to a single-stranded DNA cellulose column (15 × 2.5 cm) which had been extensively washed with Buffer B, at 10-15 ml/hr. The column was then washed with Buffer B at 20-26 ml/hr, until the optical density at 280 nm was less t h a n 0.1, and then b y the same buffer containing 0.5M NaC1. Fractions containing D N A polymerase activity were combined and dialysed against 5 litres of Buffer C (0.2 M K P O a p H 8.0, 1 mlVI EDTA, 1 mM 2-mercaptoethanol and 10% glycerol) overnight to yield Fraction I I I (30 ml). Step IV: Phosphoeellulose Chromatography. Fraction I I I was loaded at 10 ml/hr onto a column of phosphocellulose (20 × 1 cm) equilibrated with Buffer C. After washing with 20 ml of this buffer, the column was eluted with a linear gradient of 0.2-0.5 M K P O a p i t 7.5 in Buffer C (total volume 200 ml). The main D N A polymerase activity eluted at 0.3 M phosphate concentration. I n several extractions a second peak of polymerase activity was observed which represented less t h a n 2% of the total polymerase activity. This activity was analysed in the same manner as the main polymerase fraction, and although it had a different molecular weight and appeared more heat stable, it was identical in all other properties examined. I t m a y be a dissociation product of the main enzyme, or a separate enzyme, and it will not be further discussed. The fractions containing D N A polymerase activity were combined to give Fraction IVA, and stored at --20 ° C. The above purification scheme is summarised in Table 1, and the phosphocellulose chromatography elution profile is shown in Fig. 1. About a 1,350 fold purification was achieved. The apparent increase in yields routinely observed after the removal of nucleic acids (which also removed some protein) and after phosphocellulose chromatography most likely arose b y the loss of deoxyribonucleases. Fraction I V A was not homogeneous, because SDS polyacrylamide gel electrophoresis resolved several protein bands. Maximum polymerase activity required ~he presence of all four deoxyribonucleoside triphosphates, magnesium ions, KC1 and a D N A template (Table 2). Potassium ions stimulated the activity 7 fold at an optium concentration of 100 mM; sodium ions, with a similar optimum~ stimulated 4fold. Magnesium ions stimulated the activity 7 fold at an optimum concentration of 8 mM. The
Table 1. Summary of purification of DNA polymerase activities from wild-type U. maydis Fraction
Protein (rag)
I. Crude extract 3,570 IL PEG treated extract 3,140 IIL DNA-cellulose peak fractions 6 IV. Phosphocellulose peak fractions: A
3.8
Activity Specific activity Yield (units × 10-3) (units/mg protein) (%)
Purification (fold)
43 75
11 24
100 175
0 2.2
50
8,310
117
762
55
14,630
128
1,350
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P.A. Jeggo and G. R. Banks
20
.~.~.
0.5 tu
0.~
5
2O
~0 60 FRACTION NUMBER
80
Fig. 1. Phosphocellulose chromatography profile of DNA polymerase activity from wild-type U. maydis. The column was run and assays performed on fractions as described in the 'Materials and Methods' section
Table 2. Partial characterisation of Fraction IVA--requirements for reaction Reaction mixture
Template
% activity
Complete Complete Complete Complete -- DNA --KC1 --MgCI2 --dATP --dCTP + p-chloromercuribenzeate (0.16 raM)
heat denatured DNA DNase I nicked micrococcal nuclease nicked native -denatured denatured denatured denatured
100 150-300 1.2 8 < 0.2 12 16 5 3.5
denatured
1.2
Assays were carried out as described in the 'Methods' section with the calf thymus DNA templates and subtractions or additions to the standard assay mixture as indicated.
p H o p t i m u m in Tris-HC1 buffer was 7.5 w i t h less t h a n 25 % of t h e a c t i v i t y r e m a i n ing a t p H 6.5 or 9.5. 2 - M e r c a p t o e t h a n o l s t i m u l a t e d a c t i v i t y a l m o s t 2 fold a t 5 raM. H o w e v e r , t h i s was o n l y o b s e r v e d i n l a t e r e x p e r i m e n t s a n d M S H was n o t a d d e d r o u t i n e l y t o t h e a s s a y m i x t u r e . T h e a c t i v i t y was p r o p o r t i o n a l to e n z y m e c o n c e n t r a t i o n o v e r a 10 fold range, a n d its r a t e was c o n s t a n t for 50 rain. T a b l e 2 also shows t h e t e m p l a t e specificity of F r a c t i o n I V w i t h four different calf t h y m u s D N A t e m p l a t e s . T h e o r d e r of r e l a t i v e t e m p l a t e efficiencies was f o u n d t o b e DhTase I t r e a t e d > h e a t d e n a t u r e d > n a t i v e > mierococcal nuelease t r e a t e d D N A ' s . W h e n each f r a c t i o n from t h e D N A cellulose or phosphoeellulose columns was s i m u l t a n e o u s l y a s s a y e d s e p a r a t e l y w i t h t h e s e t e m p l a t e s , t h e four activities eoeluted, suggesting t h a t a single e n z y m e was responsible for t h e a c t i v i t y on all f o u r t e m p l a t e s .
DNA Polymerase Deficient Mutant of Ustilago maydls
100 '--' 80
215
- %Jeose
~50 0
cr
D 20
u')
o~
" ~"
,'~merase
INCUBAT!ONTIMEinNINUTES ~'ig. 2. Temperature sensitivity of the polymerase (.---.) and nuclease (,--,) activities of ~'raction IVA. The samples were incubated at 45° C for the times indicated when aliquots were extra~ed and assayed for polymerase or nuclease activity as described in the 'Materials and Methods' section
The molecular weight of the enzyme was determined by sedimentation in a sucrose gradient using the technique of Martin and Ames (1961). Fraction IV_& was layered onto a 5-20% sucrose gradient in 10 mM K P O 4 pI-I 7.5, 1 mM E D T A and 1 mM 2-mereaptoethanol. Cytochrome C and bovine serum albumin were used as standard markers in the same gradients. After eentrifugation in the SW 56 rotor of a Beckman L2-65B centrifuge for 16 hrs at 40,000 r p m and 4 ° C, equal drop fractions were collected and an aliquot of each assayed for polymerase activity. The positions of the marker proteins were determined b y diluting the remainder of each fraction and determining its optical densities at 420 and 280 nm respectively. The molecular weight was estimated to be 140-160,000 daltons. Deoxyribonuclease Activity. Fraction IVA had associated deoxyribonuclease activity. However, on further fractionation some separation of nuclease from polymerase activity was observed (Jeggo, 1973). Fig. 2 shows the thermal denaturation curves for the polymerase and auclease activities of Fraction IVA. The nuclease activity was relatively stable at 45 °, whilst the polymerase was more labile, suggesting t h a t a different enzyme m a y be responsible for this activity (see Discussion section).
2. Extraction and Characterization o] the DNA Polymerase /rom the Mutant pol 1-1 Since the pot 1-1 m u t a n t of U. maydis is blocked in D N A synthesis at the restrictive temperature, and has reduced levels of polymerase activity (Jeggo et aL, 1973), it was of interest to purify the enzyme from this mutant strain and compare its properties to those of Fraction IVA from the wild-type strain. pol 1-1 cells were growrt at 22 ° C and the polymerase activity purified exactly as for the wild-type strain. The elution profile of the activity from a phosphocellulose column was identical to t h a t observed for the wild-type, the minor peak of activity also being observed in this strain. The purified major fraction from the
216
P. A. Jeggo and G. R. Banks 100
80 5O
< 40
5 c~
< 2O
4
8 TIME
12 15 at 40°C {MINS)
20
Fig. 3. Temperature sensitivity of polymerase Fractions IVA (~--*) from wild-type strain and IVB (o--o) from pol 1-1 strain. The samples were incubated at 40° C for the times indicated when aliquots were extracted and assayed for polymerase activity
pol 1-1 strain was labelled Fraction IVB. Although less polymerase activity was obtained from this strain (which m a y reflect a decreased stability of the enzyme), all the characteristics of Fraction I V B were similar to those of Fraction IVA from the wild-type strain, with the exception of their heat stabilities. The purified polymerase from the pol 1-1 strain was much more heat labile t h a n t h a t from wild-type (Fig. 3), in' agreement with the evidence previously obtained t h a t the major D N A polymerase of the Tel 1-1 m u t a n t is temperature sensitive compared to t h a t of the wild-type strain (Jeggo et al., 1973). A relatively heat stable (at 40 ° C) component of Fraction I V B was observed in purified fractions, the proportion of which varied with extraction procedure and storage conditions.
3. Growth Characteristics o] pol 1-1 U. maydis normally grows in culture b y yeast-like budding of rod-shaped cells. The "pol 1-1 m u t a n t showed normal appearance at 22 ° C, but at 32°C formed filamentous cells which were mainly uninueleate (Jeggo, 1973). There was only limited loss of viability at the restrictive temperature. After 5 hrs at 32 ° C approximately 80% of the cells were viable, and after 8 hrs the viability was decreased to 50%. The growth rates of wild-type, pol 1-1 haploid and pol 1-1 homozygous and heterozygus diploid strains at varying t e m p e r a t u r e are shown in Table 3. Neither the pol 1-1 haploid nor diploid grew at 32 °, but the phenotype of the diploid is more extreme since it did not grow at 28 °. At 22 ° and 25 ° the pol 1-1 strains grew more slowly t h a n the wild-type, but there was no difference at 18 ° . The results illustrate t h a t the pol 1-1 mutation is recessive, since the heterozygous diploid was able to grow normally at the high temperature.
DNA Polymcrase Deficient ~utant of Ustilago maydis
217
Table 3. Doubling times for wild-type and pol 1-1 strains at varying temperatures Strain
Doubling time (hrs)
wild-type pol 1-1 haploid pot i-l/q- diploid pol 1-1/pol 1-1 diploid
18° C
22° C
25° C
28° C
32° C
4.4 4.4 4.4 4.4
3.0 4.3 3.0 4.1
2.3 2.9 2.3 3.4
1.9 4.1 1.9 c¢
1.5 oo 1.5 oo
0o ~ No detectable increase in cell number.
4. Genetic Segregation Previous studies showed that pol 1-1 segregates normally at meiosis and that the temperature sensitive phenotype remains associated with low DNA polymerase (Jeggo et al., 1973; P. Unrau, personal communication). However, a modifier has subsequently been discovered which has the effect of increasing growth rate and decreasing heat sensitivity (P. Unrau, personal communication).
5. Radiation Sensitivity To test whether the pol 1-1 gene product was involved in DNA repair, its sensitivity to UV and y rays was examined. The results for log phase wild-type and pol 1-1 strains incubated at 22°C are shown in Fig. 4. At low doses the m u t a n t was slightly more UV sensitive, whereas at higher doses it was slightly more resistant. Unrau (1975) has shown that the excision of pyrimidine dimers from DIqA is normal in pol 1-1 strains. The UV survial of pol 1-1 after receiving heat treatment at 32 ° C both before and after UV radiation has been examined b y P. Unrau and T. Olive (unpublished data). I n no case was there a significant enhancemant of sensitivity in comparsion with wild-type. The y-ray sensitivity of log phase cultures of wild-type and pol 1-1 was also determined. As with UV, at lower doses the m u t a n t was slightly more sensitive, whilst at higher doses it was slightly more resistant than the wild-type strain (Jeggo, 1974). The results indicate that pol 1-1 is proficient in repair of irradiated DNA.
6. Survial after NG Treatment The involvement of the pol 1-1 mutation in the repair of DNA damaged by the mutagen N-methyl-N'nitro-N-nitrosoguanidine (NG) was also examined. Fig. 5 shows the survival for wild-type and pol 1-1 strains after varying incubation times in a solution of NG. The mutant was significantly more sensitive to this treatment than the wild-type strain, but much less sensitive than various repair deficient strains (1%. Holliday, personal communication).
7. Estimation o/the Spontaneous Mutation Rate t~ecent experiments with T4 have shown that strains with altered gene 43 products (the T4 DNA polymerase) can have the properties of mutator or antimuta-
218
P.A. Jeggo and G. R. Banks 100 5C O
2(: .1C .
DNA Polymerase of Ustilago maydis: Partial Characterization of the Enzyme and a pol 1 Mutation Penelope A. Jeggo*, and Geoffrey 1~. Banks National Institute for Medical Research, Mill Hill, London EW7 tAA, England Received August 5, 1975
Summary. The major DNA polymerase activity of wild-type U. maydis has been extensively purified. It possesses a molecular weight of about 150,000 daltons and appears to require a DNA primer with a 3'-hydroxyl terminus as well as a template. The polymerase activity has also been purified from the pol 1-1 strain, which is temperature sensitive for growth and DNA synthesis, and which at the restrictive temperature contains only 10-25 % levels of the DNA polymerase activity obtained from wild-type strains. It was similar in all properties studied, except that the activity was thermolabile at 40° C compared to that from the wildtype strain. Physiological studies on the mutant showed that it was only slightly sensitive to UV, ionising radiation and nitrosoguanidine at the permissive temperature, and was proficient in genetic recombination. The results suggest that the pol 1-1 gene product does not play an important role in repair and recombination processes within the cell, and that its primary function lies in replication. Introduction DNA polymerases have now been isolated and characterised from a wide range of organisms and their tissues. Three well studied bacteria, Escherichia coli, Bacillus subtilis and Micrococcus luteus, each possess three DNA polymerases (Kornberg and Gefter, 1970, 1971, 1972; Wickuer et al., 1972; Gefter et al., 1972; Otto etal., 1973; Ganesan et al., 1973; Bazill and Gross, 1973; Hamilton, 1974), and some advances have been made towards an understanding of their role in the cell (Gefter et al., 1971; Nusslein et al., 1971; Keumpel and Yeomett, 1970; Okazaki et al., 1971 ; Gass et al., 1973 ; Hamilton et al., 1974 ; Brutlag and Kornberg, 1972). Two nuclear and a separate mitochondrial enzyme have been reported from yeast (ttelfman, 1973; Wintersberger and Wintersberger, 1970a, b), but little is known of their cellular functions. Multiple molecular weight DNA polymerases have been obtained in different intracellular locations in several mammalian systems (Chang and Bollum, 1971 ; Baril et al., 1971 ; Weissbach et al., 1971) and an attempt has been made to study their functions by estimating the variations in their levels in tissues actively involved in DNA synthesis (Chang and Bollum, 1972; Chang et al., 1973). I n general, the correlation between enzyme functions and biological processes in eukaryotic systems has depended on physiological means, since genetic techniques analogous to those using the bacterial DNA polymerase mutants have not been available.
* Present Address: Imperial Cancer Researeh Fund, Burtonhole Lane, Mill Hill, LondonNW7 1AA, England.
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P.A. Jeggo and G. R. Banks
The basidiomycete fungus, U. maydis is a yeast-like e u k a r y o t e which has been used extensively i n genetic a n d biochemical studies (Holliday, 1974; H o l l i d a y et al., 1974). The charaeterisation of several m u t a n t strains of this organism which are t e m p e r a t u r e sensitive for DlkTA synthesis has b e e n described ( U n r a u a n d Holliday, 1970). I n particular, it was shown t h a t crude extracts from t h e m u t a n t pol 1-1 c o n t a i n reduced (10-25 %) levels of D N A polymerase a c t i v i t y c o m p a r e d to those from t h e wild-type s t r a i n after i n c u b a t i o n of t h e cells a t t h e restrictive t e m p e r a t u r e . F u r t h e r m o r e , after growth at t h e permissive t e m p e r a t u r e , t h e part i a l l y purified D N A polymerase a c t i v i t y was f o u n d to be heat labile (Jeggo et al., 1973). T h e evidence suggests t h a t t h e pol 1-1 m u t a n t m a y be t e m p e r a t u r e sensitive for D N A synthesis in vivo because it possesses a t e m p e r a t u r e sensitive D N A polymerase, which is t h e replicative e n z y m e for this organism. I n this p a p e r we describe some physiological properties of this m u t a n t , a n d r e p o r t on a f u r t h e r purification a n d t h e i n i t i a l charaeterisation of t h e enzymes from t h e wild-type a n d pol 1-1 strains. A fuller a c c o u n t is g i v e n b y Jeggo (1973).
Materials a n d Methods
1. Strains Strains were obtained from stock cultures (see Holliday, 1974). Strains used were wildtype a2bl; rec 1-1 a2bl; rec 2 a~bl; uvs 3 a2bz. Various temperature sensitive strains were provided by P. Unrau. ala e and bib2 are mating type alleles, rec denotes deficiency in recombination, and uv8 sensitivity to UV light.
2. Media Cells were routinely grown in either nitrate minimal medium or complete medium as described by Itolliday (1961 a, b, 1974). For solid medium, New Zealand Davis agar was added at 20 g per litre. Mating medium consisted of 1.7 g 'Difco' cornmeal agar, 1 g Davis agar and 1 g activated charcoal added to 100 ml CM.
3. Cetl-t~ree Extracts Cells were grown in liquid complete medium to late log phase (1 × 107 cells/ml) after inoculation from a starter culture. Cell counts were made with a Coulter counter Model A after appropriate dilution. Cultures (routinely 1 litre) were harvested and washed twice in extracting buffer (0.01 phosphate pit 8.0, 0.001 M EDTA, 0.001 M MSH) at 4° C in a refrigerated centrifuge (Sorvall RC-2B) at 9,000 rpm for 5 rains. Washed cells were resuspended in 3 ml extracting buffer, half a volume of cold acid-washed Ballotini beads (no. 11) added, and the cells disintegrated by shaking in a Mikkle disintegrator for 10 rains. Beads and cellular debris were removed by ccntrifugation at 9,000 rpm for 20 rains, after which the supernatant was centrifuged 37,000 rpm for 1 hr in a Type 65 head in a Beckman L2-65B ultracentrifuge. All stages were carried out at 2-4 ° C. The supernatant from the final centrifugation was used as a crude cell-free extract.
4. Large Scale Culture Large quantities of wild-type haploid cells, strain a~bl, were grown in 300 L medium containing 3 kg Oxoid Yeast Extract, 6 kg Oxoid Bacteriological Peptone, 6 kg sucrose and 50 ml Silicone R.D. at 32° C. The cells were harvested in late log phase by centrifugation in a Sharples Super centrifuge Model 6A at 15,000 rpm, and stored as a pellet at --20 ° C. Large quantities of the mutant strain (pol 1-1) were grown overnight at 22°C to 1 × 10~ cells/ml, par~ harvested and the remainder incubated for 5 hrs at 32° C.
DNA Polymerase Deficient Mutant of Ustilago maydis
211
5. Enzymes and Substrates Pancreatic DNase (DNase 1), micrococcal DNase and calf thymus DNA were purchased from the Worthington Biochemical Corp. Phosphocellulose (P 11) was from the Whatman Co. deoxyribonucleoside triphosphates from PL Biochemieals Inc. and [8tt]-thymidine and [aH]d T T P (11.2 Ci/mM) from the Radiochemical Centre.
6. D N A Polymerase Assay The reaction measures the incorporation of [aH]-dTTP into acid insoluble DNA. The mixture contained 10 ~mole Tris-HC1 p H 7.5, 5 nmole each of dATP, dCTP and dGTP, 0.5 nmole dTTP, 0.1 nmole [3H]-dTTP (1.25 ~zCi), 20 ~zmole KCI, 1.5 fxmole MgCl~, 75 ~xg heat denatured calf thymus DNA and aliquots of the enzyme in a total volume of 0.15 ml. After incubation at 32 ° C for 40 rain, 0.1 ml of the mixture was pipetted onto a Whatman 3 mm filter paper square, which was immediately immersed in cold 5 % TCA containing 1% sodium pyrophosphato. The filters were washed in two further changes of TCA and finally in cold 95 % ethanol, dried and counted in scintillation fluid containing 5 g 2,5-diphenyloxazole (PPO) and 0.2 g 1,4-bis (2-4-methyl-5-phenyloxazolyl) benzene (dimethyl POPOP) per litre of toluene. One unit of activity is defined as the amount of protein which incorporates 1 pmole of dTTP into acid insoluble DNA under the above conditions.
7. Deoxyribonuclease Assay The method measures the release of acid soluble radioactivity from E. coli [aH]-DNA. The mixture contained 55 fzmole Tris-tIC1 pit 7.2, 0.15 ~mole MgC12, 5 ~g E. coli [3It]-DNA and enzyme in a volume of 0.15 ml. After incubation at 32 ° C for 40 rains, 0.1 ml (1.5 mg/ml) carrier DNA and 0.2 ml cold 5% TCA were added, and after holding in ice for 10 rains, the tubes were centrifuged at 10,000 rpm for 10 rains. 0.1 ml of the supernatant solution was counted in scintillation fluid containing 60 g naphthalene, 4 g PPO, 0.2 g dimethyl P O P O P 100 ml methanol and 900 ml 1.4 dioxan.
8. DIYA Calf thymus DNA was dissolved at 3 mg/ml in 0.2 M Tris-IiC1 p i t 8.0-0.02 M NaC1. I t was denatured by incubation in a boiling water bath for 10 rains, followed by rapid cooling in ice. DNase I nicked calf thymus DNA was prepared as described by Aposhian and Kornberg (1962), and mierococcal nuclease nicked DNA by the method of Richardson and Kornberg (1964). [aHJ-thymidine labelled E. coli DNA was extracted by iVIarmur's method (1961).
9. Other Biochemical Methods Single-stranded calf thymus DNA cellulose was prepared as described by Alberts and Herrick (1971) using Munktell 410 cellulose. About 1.5 mg DNA/g cellulose remained bound after extensive washing. Protein concentrations were determined by the method of Lowry et al. (1951) using bovine serum albumin as the standard.
10. Growth Rates Cells were grown in liquid CM with vigorous aeration. Cell counts were made with a Coulter counter.
11. Irradiation UV irradiation was from a Hanovia low-pressure germicidal lamp delivering 46 ergs per mm 2 per second at a distance of 15 cm. Cells were treated after spreading on CM, or in suspension in sterile water, at l0 s cells/ml. Precautions were taken to prevent photoreactivation.
212
P . A . Jeggo and G. R. Banks
F-irradiation was from a Gammabeam 650 Co60 source delivering 40 Krads per rain to the cell suspension. Cells were irradiated at 107/ml in a beaker with stirring.
12. Treatment with N-Methyl N Nitrosoguanidine (NG) Log phase cells were grown in CM at 22 ° C and NG added to a final concentration of 200 ~g/ml. The culture was incubated with shaking at 22 ° C and at 6 rain intervals samples were taken and diluted immediately 100 fold to prevent further mutagenic action. Suitable dilutions were plated on CM and the survival estimated after 5-8 days incubation at 22 ° C.
13. Isolation of Diploids Diploids were isolated by mixing strains of complementary mating types on mating medium, each parent having a different nitritional requirement. When white hyphae had appeared, the plate was replica plated to ~ on which heterokaryons grew very slowly, whereas diploids grew vigorously. Single colony isolates were made of each diploid.
ld. Spontaneous Mutation The spontaneous mutation frequency was measured by a fluctuation test using the method of the median developed by Lea and Coulson (1949). For each strain, 10 tubes each conraining 5 ml CM were inoculated with 103 cells/ml from a log phase starter culture. The parallel cultures were grown for 2-3 days at 22 ° C to stationary phase and suitable dilutions plated as required.
Results
1. Puri/ication o] DNA Polymerase A l l o p e r a t i o n s were c a r r i e d o u t a t 4 ° C a n d c e n t r i f u g a t i o n was a t 9,000 r p m for 20 rains unless o t h e r w i s e i n d i c a t e d . Step I: Preparation o/Crude Extract. 300 g frozen cells were t h a w e d a n d w a s h e d in Buffer A (10 m M Tris-HC1 p H 8.0, 1 m M 2 - m e r e a p t o e t h a n o l a n d 10% glycerol). A f t e r centrifugation, t h e cells were r e s u s p e n d e d in 300 m l Buffer A a n d 600 g cooled a c i d - w a s h e d B a i l o t i n i No. 11 glass b e a d s a d d e d . T h e ceils were disinteg r a t e d in a cooled W a r i n g B l e n d e r u n t i l a b o u t 100% cell b r e a k a g e was o b s e r v e d u n d e r t h e microscope. T h e t e m p e r a t u r e was m a i n t a i n e d below 15 ° C. A f t e r allowing t h e b e a d s t o settle, t h e s u p e m a t a n t was d e c a n t e d a n d t h e d e b r i s w a s h e d twice w i t h 100 m l of Buffer A. T h e r e s u l t i n g s u p e r n a t a n t s were c o m b i n e d a n d c e n t r i f u g e d t o give F r a c t i o n I (170 ml). Step II: Removal o] Nucleic Acids. Nucleic acids were r e m o v e d b y t h e p h a s e s e p a r a t i o n t e c h n i q u e of A l b e r t s a n d H e r r i c k (1971). Solid NaC1 was a d d e d slowly t o s t i r r e d F r a c t i o n I t o a c o n c e n t r a t i o n of 1.7 M. A 30% p o l y e t h y l e n e glycerol ( C a r b o w a x 6000) s o l u t i o n was a d d e d t o a final c o n c e n t r a t i o n of 10% a n d t h e suspension s t i r r e d for 30 rain in ice. A f t e r c e n t r i f u g a t i o n a t 10,000 r p m for 15 mills, t h e s u p e r n a t a n t solution was d i a l y s e d a g a i n s t Buffer B (10 m M Tris-HC1 p t I 8.0, I m M E D T A , 1 m M 2 - m e r c a p t o e t h a n o l , 5 0 m M N a C 1 a n d 10% glycerol) (51itre w i t h 2 changes). F i n a l l y , t h e e x t r a c t was clarified b y c e n t r i f u g a t i o n a t 37,000 r p m in a T y p e 40 r o t o r on a B e c k m a n L2-65B centrifuge for 60 mins t o give T r a c t i o n I I (300 ml). Step III: DNA-cellulose Chromatography. I t h a d p r e v i o u s l y b e e n shown t h a t D N A p o l y m e r a s e a c t i v i t y f r o m U. maydis e l u t e d f r o m a DNA-eellulose c o l u m n
DNA Polymerase Deficient Mutant of Ustilagomaydis
213
at 0.dM NaC1. To facilitate the large scale preparation of purified D N A polymerase, the enzyme was eluted from the DNA-cellulose column in a stepwise manner. Fraction I I was applied to a single-stranded DNA cellulose column (15 × 2.5 cm) which had been extensively washed with Buffer B, at 10-15 ml/hr. The column was then washed with Buffer B at 20-26 ml/hr, until the optical density at 280 nm was less t h a n 0.1, and then b y the same buffer containing 0.5M NaC1. Fractions containing D N A polymerase activity were combined and dialysed against 5 litres of Buffer C (0.2 M K P O a p H 8.0, 1 mlVI EDTA, 1 mM 2-mercaptoethanol and 10% glycerol) overnight to yield Fraction I I I (30 ml). Step IV: Phosphoeellulose Chromatography. Fraction I I I was loaded at 10 ml/hr onto a column of phosphocellulose (20 × 1 cm) equilibrated with Buffer C. After washing with 20 ml of this buffer, the column was eluted with a linear gradient of 0.2-0.5 M K P O a p i t 7.5 in Buffer C (total volume 200 ml). The main D N A polymerase activity eluted at 0.3 M phosphate concentration. I n several extractions a second peak of polymerase activity was observed which represented less t h a n 2% of the total polymerase activity. This activity was analysed in the same manner as the main polymerase fraction, and although it had a different molecular weight and appeared more heat stable, it was identical in all other properties examined. I t m a y be a dissociation product of the main enzyme, or a separate enzyme, and it will not be further discussed. The fractions containing D N A polymerase activity were combined to give Fraction IVA, and stored at --20 ° C. The above purification scheme is summarised in Table 1, and the phosphocellulose chromatography elution profile is shown in Fig. 1. About a 1,350 fold purification was achieved. The apparent increase in yields routinely observed after the removal of nucleic acids (which also removed some protein) and after phosphocellulose chromatography most likely arose b y the loss of deoxyribonucleases. Fraction I V A was not homogeneous, because SDS polyacrylamide gel electrophoresis resolved several protein bands. Maximum polymerase activity required ~he presence of all four deoxyribonucleoside triphosphates, magnesium ions, KC1 and a D N A template (Table 2). Potassium ions stimulated the activity 7 fold at an optium concentration of 100 mM; sodium ions, with a similar optimum~ stimulated 4fold. Magnesium ions stimulated the activity 7 fold at an optimum concentration of 8 mM. The
Table 1. Summary of purification of DNA polymerase activities from wild-type U. maydis Fraction
Protein (rag)
I. Crude extract 3,570 IL PEG treated extract 3,140 IIL DNA-cellulose peak fractions 6 IV. Phosphocellulose peak fractions: A
3.8
Activity Specific activity Yield (units × 10-3) (units/mg protein) (%)
Purification (fold)
43 75
11 24
100 175
0 2.2
50
8,310
117
762
55
14,630
128
1,350
214
P.A. Jeggo and G. R. Banks
20
.~.~.
0.5 tu
0.~
5
2O
~0 60 FRACTION NUMBER
80
Fig. 1. Phosphocellulose chromatography profile of DNA polymerase activity from wild-type U. maydis. The column was run and assays performed on fractions as described in the 'Materials and Methods' section
Table 2. Partial characterisation of Fraction IVA--requirements for reaction Reaction mixture
Template
% activity
Complete Complete Complete Complete -- DNA --KC1 --MgCI2 --dATP --dCTP + p-chloromercuribenzeate (0.16 raM)
heat denatured DNA DNase I nicked micrococcal nuclease nicked native -denatured denatured denatured denatured
100 150-300 1.2 8 < 0.2 12 16 5 3.5
denatured
1.2
Assays were carried out as described in the 'Methods' section with the calf thymus DNA templates and subtractions or additions to the standard assay mixture as indicated.
p H o p t i m u m in Tris-HC1 buffer was 7.5 w i t h less t h a n 25 % of t h e a c t i v i t y r e m a i n ing a t p H 6.5 or 9.5. 2 - M e r c a p t o e t h a n o l s t i m u l a t e d a c t i v i t y a l m o s t 2 fold a t 5 raM. H o w e v e r , t h i s was o n l y o b s e r v e d i n l a t e r e x p e r i m e n t s a n d M S H was n o t a d d e d r o u t i n e l y t o t h e a s s a y m i x t u r e . T h e a c t i v i t y was p r o p o r t i o n a l to e n z y m e c o n c e n t r a t i o n o v e r a 10 fold range, a n d its r a t e was c o n s t a n t for 50 rain. T a b l e 2 also shows t h e t e m p l a t e specificity of F r a c t i o n I V w i t h four different calf t h y m u s D N A t e m p l a t e s . T h e o r d e r of r e l a t i v e t e m p l a t e efficiencies was f o u n d t o b e DhTase I t r e a t e d > h e a t d e n a t u r e d > n a t i v e > mierococcal nuelease t r e a t e d D N A ' s . W h e n each f r a c t i o n from t h e D N A cellulose or phosphoeellulose columns was s i m u l t a n e o u s l y a s s a y e d s e p a r a t e l y w i t h t h e s e t e m p l a t e s , t h e four activities eoeluted, suggesting t h a t a single e n z y m e was responsible for t h e a c t i v i t y on all f o u r t e m p l a t e s .
DNA Polymerase Deficient Mutant of Ustilago maydls
100 '--' 80
215
- %Jeose
~50 0
cr
D 20
u')
o~
" ~"
,'~merase
INCUBAT!ONTIMEinNINUTES ~'ig. 2. Temperature sensitivity of the polymerase (.---.) and nuclease (,--,) activities of ~'raction IVA. The samples were incubated at 45° C for the times indicated when aliquots were extra~ed and assayed for polymerase or nuclease activity as described in the 'Materials and Methods' section
The molecular weight of the enzyme was determined by sedimentation in a sucrose gradient using the technique of Martin and Ames (1961). Fraction IV_& was layered onto a 5-20% sucrose gradient in 10 mM K P O 4 pI-I 7.5, 1 mM E D T A and 1 mM 2-mereaptoethanol. Cytochrome C and bovine serum albumin were used as standard markers in the same gradients. After eentrifugation in the SW 56 rotor of a Beckman L2-65B centrifuge for 16 hrs at 40,000 r p m and 4 ° C, equal drop fractions were collected and an aliquot of each assayed for polymerase activity. The positions of the marker proteins were determined b y diluting the remainder of each fraction and determining its optical densities at 420 and 280 nm respectively. The molecular weight was estimated to be 140-160,000 daltons. Deoxyribonuclease Activity. Fraction IVA had associated deoxyribonuclease activity. However, on further fractionation some separation of nuclease from polymerase activity was observed (Jeggo, 1973). Fig. 2 shows the thermal denaturation curves for the polymerase and auclease activities of Fraction IVA. The nuclease activity was relatively stable at 45 °, whilst the polymerase was more labile, suggesting t h a t a different enzyme m a y be responsible for this activity (see Discussion section).
2. Extraction and Characterization o] the DNA Polymerase /rom the Mutant pol 1-1 Since the pot 1-1 m u t a n t of U. maydis is blocked in D N A synthesis at the restrictive temperature, and has reduced levels of polymerase activity (Jeggo et aL, 1973), it was of interest to purify the enzyme from this mutant strain and compare its properties to those of Fraction IVA from the wild-type strain. pol 1-1 cells were growrt at 22 ° C and the polymerase activity purified exactly as for the wild-type strain. The elution profile of the activity from a phosphocellulose column was identical to t h a t observed for the wild-type, the minor peak of activity also being observed in this strain. The purified major fraction from the
216
P. A. Jeggo and G. R. Banks 100
80 5O
< 40
5 c~
< 2O
4
8 TIME
12 15 at 40°C {MINS)
20
Fig. 3. Temperature sensitivity of polymerase Fractions IVA (~--*) from wild-type strain and IVB (o--o) from pol 1-1 strain. The samples were incubated at 40° C for the times indicated when aliquots were extracted and assayed for polymerase activity
pol 1-1 strain was labelled Fraction IVB. Although less polymerase activity was obtained from this strain (which m a y reflect a decreased stability of the enzyme), all the characteristics of Fraction I V B were similar to those of Fraction IVA from the wild-type strain, with the exception of their heat stabilities. The purified polymerase from the pol 1-1 strain was much more heat labile t h a n t h a t from wild-type (Fig. 3), in' agreement with the evidence previously obtained t h a t the major D N A polymerase of the Tel 1-1 m u t a n t is temperature sensitive compared to t h a t of the wild-type strain (Jeggo et al., 1973). A relatively heat stable (at 40 ° C) component of Fraction I V B was observed in purified fractions, the proportion of which varied with extraction procedure and storage conditions.
3. Growth Characteristics o] pol 1-1 U. maydis normally grows in culture b y yeast-like budding of rod-shaped cells. The "pol 1-1 m u t a n t showed normal appearance at 22 ° C, but at 32°C formed filamentous cells which were mainly uninueleate (Jeggo, 1973). There was only limited loss of viability at the restrictive temperature. After 5 hrs at 32 ° C approximately 80% of the cells were viable, and after 8 hrs the viability was decreased to 50%. The growth rates of wild-type, pol 1-1 haploid and pol 1-1 homozygous and heterozygus diploid strains at varying t e m p e r a t u r e are shown in Table 3. Neither the pol 1-1 haploid nor diploid grew at 32 °, but the phenotype of the diploid is more extreme since it did not grow at 28 °. At 22 ° and 25 ° the pol 1-1 strains grew more slowly t h a n the wild-type, but there was no difference at 18 ° . The results illustrate t h a t the pol 1-1 mutation is recessive, since the heterozygous diploid was able to grow normally at the high temperature.
DNA Polymcrase Deficient ~utant of Ustilago maydis
217
Table 3. Doubling times for wild-type and pol 1-1 strains at varying temperatures Strain
Doubling time (hrs)
wild-type pol 1-1 haploid pot i-l/q- diploid pol 1-1/pol 1-1 diploid
18° C
22° C
25° C
28° C
32° C
4.4 4.4 4.4 4.4
3.0 4.3 3.0 4.1
2.3 2.9 2.3 3.4
1.9 4.1 1.9 c¢
1.5 oo 1.5 oo
0o ~ No detectable increase in cell number.
4. Genetic Segregation Previous studies showed that pol 1-1 segregates normally at meiosis and that the temperature sensitive phenotype remains associated with low DNA polymerase (Jeggo et al., 1973; P. Unrau, personal communication). However, a modifier has subsequently been discovered which has the effect of increasing growth rate and decreasing heat sensitivity (P. Unrau, personal communication).
5. Radiation Sensitivity To test whether the pol 1-1 gene product was involved in DNA repair, its sensitivity to UV and y rays was examined. The results for log phase wild-type and pol 1-1 strains incubated at 22°C are shown in Fig. 4. At low doses the m u t a n t was slightly more UV sensitive, whereas at higher doses it was slightly more resistant. Unrau (1975) has shown that the excision of pyrimidine dimers from DIqA is normal in pol 1-1 strains. The UV survial of pol 1-1 after receiving heat treatment at 32 ° C both before and after UV radiation has been examined b y P. Unrau and T. Olive (unpublished data). I n no case was there a significant enhancemant of sensitivity in comparsion with wild-type. The y-ray sensitivity of log phase cultures of wild-type and pol 1-1 was also determined. As with UV, at lower doses the m u t a n t was slightly more sensitive, whilst at higher doses it was slightly more resistant than the wild-type strain (Jeggo, 1974). The results indicate that pol 1-1 is proficient in repair of irradiated DNA.
6. Survial after NG Treatment The involvement of the pol 1-1 mutation in the repair of DNA damaged by the mutagen N-methyl-N'nitro-N-nitrosoguanidine (NG) was also examined. Fig. 5 shows the survival for wild-type and pol 1-1 strains after varying incubation times in a solution of NG. The mutant was significantly more sensitive to this treatment than the wild-type strain, but much less sensitive than various repair deficient strains (1%. Holliday, personal communication).
7. Estimation o/the Spontaneous Mutation Rate t~ecent experiments with T4 have shown that strains with altered gene 43 products (the T4 DNA polymerase) can have the properties of mutator or antimuta-
218
P.A. Jeggo and G. R. Banks 100 5C O
2(: .1C .
