CurrentGenetics (1984)8:19-21
Current Genetics © Springer-Verlag 1984
Identification of a mitochondrial endonuclease involved in mt-DNA repair of Saccharomyces cerevisiae* C. V. Lusena and C. Champagne
Molecular Genetics Section, Division of Biological Sciences, National Research Council of Canada, Ottawa, Ontario Canada K1A 0R6
Summary. The activity of a yeast mitochondrial endonuclease extracted from mutants (sasl) with increased sensitivity to petite-inducing treatments was compared to that from wild-type cells. The specificity of the endonuclease was altered in haploids carrying a single mutant nuclear gene that conferred increased sensitivity to petite induction by ultraviolet light, by growth at an elevated temperature and by growth in the presence of aminopterin and sulfanilamide. At high ionic strengths the endonuclease from the mutants digested double stranded DNA much faster than did that from the wild-type strain. Also the mutant enzyme was less selective for poly(dA) • poly(dU) in comparison to poly(dA) • poly(dT); it had less preference for reduced hydrogen bond strength between the strands of double stranded DNA. Results indicate that this endonuclease, as a degrading enzyme, is involved in the initial step in repair of damaged mitochondrial DNA. Key words: mt-endonuclease - mt-DNA repair - Petiteinduction
Introduction
Both the remodelling of mt-DNA observed in petite production by yeast and the repair processes observed in mitochondria during liquid holding after ultraviolet irradiation require the intervention of a nuclease (Heude and Moustacchi t979). One candidate for this function is a recently-isolated endonuclease that preferentially cuts both strands of double-stranded DNA at regions of
* This paper is NRCC Publication No. 21169 Offprint requests to." C. V. Lusena
reduced hydrogen-bond strength (Morosoli and Lusena 1982). If this endonuclease is indeed involved in repair, then mutations affecting its activity might be expected to confer sensitivity to any petite-inducing treatment which acts by reducing locally the stability of the double strand of DNA. Such treatments include the presence of sulfanilamide plus aminopterin in the medium (Barclay and Little 1978), exposure to ultraviolet irradiation (Moustacchi et al. 1975), and growth at the elevated temperature of 36 °C (Schenberg-Frascino and Moustacchi 1972). Individual mutants that display this multiple sensitivity have now been isolated and shown to be a consequence of recessive nuclear mutations in one gene (Lusena and James 1982). In this report we compare the in vitro activity of the endonucleases from these mutants with that from the wild-type strain. The enzyme from the mutants was altered in the expected way; it was less selective for DNA regions with lower hydrogen-bond strength, i.e. improper pairings. Therefore, the mutated gene that confers multiple sensitivity to petite-inducing treatments code for one of the proteins in the mitochondrial endonuclease complex. It is concluded that this endonuclease is a degrading enzyme involved in the initial step of the mitochondrial repair mechanism.
Materials and methods Preparation o f endonuclease. Four strains of Saccharomyces eerevisiae, C634-1A (SAS), #22 (sasl-1), #35 (sasl-2), #34 (sas2-1)
(Lusena and James 1982) were grown in lactate medium to late log phase. From these cells mitochondrial fractionswere prepared by a method described preciously (Morosoli and Lusena 1980) and washed once in 0.5 M Sorbitol. The pellets were frozen and stored at -20 °C until used. The endonuclease was prepared as described by Morosoli and Lusena (1980) except that the linear gradients were replaced by step elution. For hydroxylapatite
20
C.V. Lusena and C. Champagne: Yeast endonuclease involved in mt-DNA repair
Table 1. Petites (percent a) induced in strains with multiple sensitivity by sulfanilamide-aminopterin treatment, by UV irradiation and by growth at an elevated temperature Lab #
Genotype
Control
SAb
C634-1A 22 35 34
SAS sasl-1 sasl-2 sas2-1
4.3 6.0 3.1 6.2
4.9 98.4 99.2 79.6 d
36 °C
UV c
3.9 76.7 87.6 10.0
5.1 20.2 12.3 80.7
a At least 1,000 colonies were scored on YPDG plates b After 24 h growth in the presence of 3 #g aminopterin and 150 #g sulfanilamide/ml c UV irradiation 750 ergs, t x 106 cells/ml in saline, survival was between 10 and 20% d After 72 h growth in the presence of drugs
Table 2. Comparison of the endonuclease activity a of the mutants at various ionic strangths Genotype
Ionic Strengt hb 0.05
SAS sasl-1
sasl-2 sas2-1
24.6 19.7 20.0 22.8
0.10 ± 1.4 ± 0.9 ± 2.2 -+0.7
16.9 21.2 21.5 12.8
0.15 ± 0.5 -+0.6 +- 1.1 -+ 1.3
7.3 22.3 14.4 6.0
0.20 ± 0.8 +- 1.6 ± 1.1 ± 0.8
1.6 8.8 6.2 1.5
± 0.3 -+ 0.7 ± 0.5 ± 0.4
a Acid-soluble fragments from [methyl-3H] thymidine labelled M)NA at 30 °C expressed as percent of the amount released from denatured M)NA. Averages and standard errors of four determinations with two enzyme preparations Contributed by 0.01 M MgC12 (ionic strength 0.02), 0.01 M Tris-HC1 buffer pH 7.5 (ionic strength 0.01) and, the remainder, by KC1
chromatography the eluent was 0.I5 M potassium phosphate buffer pH 7.0, 0.25 M KC1, 1% Triton X-100. For hepaxinagarose chromatography the eluent was 0.35 M KC1, 0.01 M TrisHC1 buffer ph 7.5, 1% Triton X-100 (sic). Only the fractions with very high activity were pooled in order to avoid having to concentrate the sample. The sample was dialysed against 0.1 M KC1, 0.01 M Tris-HC1 and 1% Triton Xq00. With 1% Triton the enzyme solutions remained clear for several months in the refrigerator with no change in the activity. This endonuclease preparation is a stable complex, dissociated by SDS and resolved by polyacrylamide gel electrophoresis in four proteins (Morosoli and Lusena 1980).
Assays of endonucleases. The release of acid-soluble fragments from h-DNA labelled with (methyl-3H) thymine was measured by a method already described (Morosoli and Lusena 1982). The amount of nucleotides released was proportional to enzyme concentration and digestion time. Various ionic strengths were obtained by changing the KC1 concentrations. The endonuclease preparations were calibrated with denatured h-DNA at 0.08 ionic strength. In the range under study, ionic strength had no effect on the activity with denatured DNA. All strains yielded compa-
table amounts of endonuclease activity with denatured DNA. The results with native h-DNA were expressed as percent of the activity with denatured X-DNA and, therefore, when divided by 100, represent nmol of nucleotide released per min per enzyme unit (Morosoli and Lusena 1980). The release of acid soluble fragments from poly(dA)poly(dT) and poly(dA) • poly(dU) (P-L. Biochemicals) at 30 °C and 0.15 M ionic strength was measured spectrophotometrically (Morosoli and Lusena 1982). The endonuclease activity was expressed as percent of the activity with denatured X-DNA.
Results All three m u t a n t strains investigated had d e m o n s t r a t e d an increased sensitivity to petite-induction b y exposure to sulfanilamide-aminopterin with a c o n c o m i t a n t increase in petite p r o d u c t i o n after U V irradiation and after growth at an elevated t e m p e r a t u r e (Lusena and James 1982). For two o f these strains, # 2 2 and # 3 5 , genetic analyses d e m o n s t r a t e d that multiple sensitivity was attributable to n o n - c o m p l e m e n t i n g recessive m u t a t i o n s at a single locus (sasl-1 and sasl-2). For one strain # 3 4 , gegetic analyses d e m o n s t r a t e d the presence o f a recessive m u t a t i o n in another locus, sas2-1, conferring delayed sensitivity to sulfanilamide-aminopterin, U V sensitivity, b u t little or no sensitivity t o elevated t e m p e r a t u r e . Subsequent tests to compare endonuclease activities were carried out with segregants o f second-generation backcrosses o f the three strains genetically analysed ( # 2 2 , # 3 5 and # 3 4 ) . Table 1 presents data concerning the sensitivity o f these segregants to petite-inducing treatments. Multiple sensitivity has been retained b y # 2 2 (sasl-1)and b y # 3 5 (sasl-2),but # 3 4 (sas2-1)has lost its sensitivity to elevated t e m p e r a t u r e and shows sensitivity to drugs only after 72 h. Endonuclease activities were compared at four ionic strengths and the results are summarised in Table 2. The activity o f the wild t y p e e n z y m e decreased sharply w i t h increased ionic strength. In contrast the e n z y m e activities o f sasl-1 and sasl-2 were m u c h less affected b y changes in ionic strength. For the m u t a n t sas2-1 the enz y m e activity was not significantly different f r o m the wild-type. Clearly the t w o alMic m u t a t i o n s in the SAS1 gene resulted in an increased activity o f the m i t o c h o n d r i a l endonuclease with double stranded D N A at high ionic strength, i. e. w h e n the b o n d strength b e t w e e n strands was increased. M u t a t i o n in the SAS2 gene did n o t affect the e n z y m e activity. The abilities o f the wild-type and m u t a n t endonuclease to act on p o l y ( d A ) " p o l y ( d U ) and p o l y ( d A ) - p o l y ( d T ) , and to discriminate b e t w e e n p o l y ( d U ) and p o l y ( d T ) are shown in Table 3. The e n z y m e s isolated f r o m the sasl m u t a n t s have a m u c h higher activity w i t h b o t h substrates t h a n e n z y m e s f r o m wild t y p e cells. Their ability to discriminate b e t w e e n p o l y ( d T ) and p o l y ( d U ) is indicated
C. V. Lusena and C. Champagne: Yeast endonuclease involved in mt-DNA repair Table 3. Comparison of the endonuclease activitya of the mutants with polydA-polydU and polydA-polydT as substrate at 0.15 ionic strength Genotype
SAS sasl-1 sasl-2 sas2-1
Substrate
Ratio
AU
AT
16.2 36.4 21.9 10.5
3.4 t4.6 8.9 2.5
AU/AT 4.8 2.5 b 2.5 b 4.1
a Acid-soluble fragments released from synthetic polynucleotides at 30 °C expressed as percent of the amount released from denatured XDNA. Averages for two enzyme preparations b Values different from wild type at 1% level of significance
by the AU/AT ratio. These ratios show that the preparations from sasl mutants were not as discriminating as those from either the wild type or the sas2 mutant.
Discussion It is clear that the specificity o f the mitochondrial endonuctease is altered b y the presence o f either of two mutations, sasl-1, and sasl-2, in the SAS1 gene. These mutant enzyme activities are more active with doublestranded DNA than the enzyme from wild type cells when the hydrogen-bond strength between the strands is increased b y ionic strength or b y base composition. Therefore, the mutant enzyme activities are less able to discriminate between legitimate and mismatch base pairs. It is also clear that this loss of specificity is accompanied b y an increase in sensitivity of the mutant cells to induction o f petites b y exposure to sulfanilamide-aminopterin, ultraviolet irradiation, and elevated temperatures. All these treatments locally weaken the stability o f the double strand o f DNA. The mutant enzyme is not able to cope with these changes as efficiently as the normal enzyme. What is the role o f the mitochondrial endonuclease? We propose that the endonuclease under study in this report normally is responsible for the cuts necessary to eliminate mismatched regions in the mt-DNA. The normal enzyme in the wild-type strain can correct the errors induced b y the treatments described in this report, while the mutant enzyme cannot. After ultraviolet irradiation an extensive breakdown o f mt-DNA occurs (Moustacchi
21
et al. 1975) with selective degradation of A-T rich regions (Hixon and Moustacchi 1978). Similar degradation occurs after treatment with high concentrations o f sulfanilamide and aminopterin (Barclay and Little 1978). Both treatments yield petites. The remodelled DNA o f the resulting petites contains large deletions, rearrangements and repetitions. The endonuclease is responsible for the initial cuts, which are followed b y recombination and ligation (Gaillard et al. 1980). The role o f the endonuclease is therefore to perform the first step o f the repair mechanism o f mt-DNA and, when this repair is not performed adequately, it becomes part o f the process of petite production. Nucleases located in the nucleus and involved in the repair mechanisms for the removal o f mismatched segments are very specific (i. e. removal of thymidine dimers): a great deal of precision is required to protect the unique genome of each cell. Evidence for such enzymes has not been found in yeast mitochondria (Prakash 1975). Perhaps they are not necessary because there are many genomes in each cell. The mitochondrial endonuclease has a more general function: it preferentially cuts any region with lower attraction between the DNA strands. The process must be far from error-free, even under normal conditions, considering the high sensitivity o f yeast to petite induction. Acknowledgements. We thank Rosemary O'Connell for the data in Table 1 and Dr. A. P. James for discussions and criticisms of the manuscript.
References Barclay BJ, Little JG (1978) Mol Gen Genet 160:33-40 Gaillard C, Strauss F, Bernardi G (1980) Nature 283:218-220 Hixon S, Moustacchi E (1978) Biochem Biophys Res Commun 81:288-296 Heude M, Moustacchi E (1979) Genetics 93:81-103 Lusena CV, James AP (1982) Can J Genet Cyto124:781-786 Morosoli R, Lusena CV (1980) Eur J Biochem 110:431-437 Morosoli R, Lusena CV (1982) Can J Biochem 60:757-762 Moustacchi E, Waters R, Heude M, Chanet R (1975) In: Nygaard OF, Adler HI, Sinclair WK (eds) Radiation research, biomedical, chemical and physical perspectives. Academic Press, New York, pp 632 650 Prakash L (1975) J Mol Bio198:781-795 Schenberg-Frascino A, Moustacchi E (1972) Mol Gen Genet 115:243-257 Communicated b y R. J. Schweyen Received April 15 / July 4, 1983
Current Genetics © Springer-Verlag 1984
Identification of a mitochondrial endonuclease involved in mt-DNA repair of Saccharomyces cerevisiae* C. V. Lusena and C. Champagne
Molecular Genetics Section, Division of Biological Sciences, National Research Council of Canada, Ottawa, Ontario Canada K1A 0R6
Summary. The activity of a yeast mitochondrial endonuclease extracted from mutants (sasl) with increased sensitivity to petite-inducing treatments was compared to that from wild-type cells. The specificity of the endonuclease was altered in haploids carrying a single mutant nuclear gene that conferred increased sensitivity to petite induction by ultraviolet light, by growth at an elevated temperature and by growth in the presence of aminopterin and sulfanilamide. At high ionic strengths the endonuclease from the mutants digested double stranded DNA much faster than did that from the wild-type strain. Also the mutant enzyme was less selective for poly(dA) • poly(dU) in comparison to poly(dA) • poly(dT); it had less preference for reduced hydrogen bond strength between the strands of double stranded DNA. Results indicate that this endonuclease, as a degrading enzyme, is involved in the initial step in repair of damaged mitochondrial DNA. Key words: mt-endonuclease - mt-DNA repair - Petiteinduction
Introduction
Both the remodelling of mt-DNA observed in petite production by yeast and the repair processes observed in mitochondria during liquid holding after ultraviolet irradiation require the intervention of a nuclease (Heude and Moustacchi t979). One candidate for this function is a recently-isolated endonuclease that preferentially cuts both strands of double-stranded DNA at regions of
* This paper is NRCC Publication No. 21169 Offprint requests to." C. V. Lusena
reduced hydrogen-bond strength (Morosoli and Lusena 1982). If this endonuclease is indeed involved in repair, then mutations affecting its activity might be expected to confer sensitivity to any petite-inducing treatment which acts by reducing locally the stability of the double strand of DNA. Such treatments include the presence of sulfanilamide plus aminopterin in the medium (Barclay and Little 1978), exposure to ultraviolet irradiation (Moustacchi et al. 1975), and growth at the elevated temperature of 36 °C (Schenberg-Frascino and Moustacchi 1972). Individual mutants that display this multiple sensitivity have now been isolated and shown to be a consequence of recessive nuclear mutations in one gene (Lusena and James 1982). In this report we compare the in vitro activity of the endonucleases from these mutants with that from the wild-type strain. The enzyme from the mutants was altered in the expected way; it was less selective for DNA regions with lower hydrogen-bond strength, i.e. improper pairings. Therefore, the mutated gene that confers multiple sensitivity to petite-inducing treatments code for one of the proteins in the mitochondrial endonuclease complex. It is concluded that this endonuclease is a degrading enzyme involved in the initial step of the mitochondrial repair mechanism.
Materials and methods Preparation o f endonuclease. Four strains of Saccharomyces eerevisiae, C634-1A (SAS), #22 (sasl-1), #35 (sasl-2), #34 (sas2-1)
(Lusena and James 1982) were grown in lactate medium to late log phase. From these cells mitochondrial fractionswere prepared by a method described preciously (Morosoli and Lusena 1980) and washed once in 0.5 M Sorbitol. The pellets were frozen and stored at -20 °C until used. The endonuclease was prepared as described by Morosoli and Lusena (1980) except that the linear gradients were replaced by step elution. For hydroxylapatite
20
C.V. Lusena and C. Champagne: Yeast endonuclease involved in mt-DNA repair
Table 1. Petites (percent a) induced in strains with multiple sensitivity by sulfanilamide-aminopterin treatment, by UV irradiation and by growth at an elevated temperature Lab #
Genotype
Control
SAb
C634-1A 22 35 34
SAS sasl-1 sasl-2 sas2-1
4.3 6.0 3.1 6.2
4.9 98.4 99.2 79.6 d
36 °C
UV c
3.9 76.7 87.6 10.0
5.1 20.2 12.3 80.7
a At least 1,000 colonies were scored on YPDG plates b After 24 h growth in the presence of 3 #g aminopterin and 150 #g sulfanilamide/ml c UV irradiation 750 ergs, t x 106 cells/ml in saline, survival was between 10 and 20% d After 72 h growth in the presence of drugs
Table 2. Comparison of the endonuclease activity a of the mutants at various ionic strangths Genotype
Ionic Strengt hb 0.05
SAS sasl-1
sasl-2 sas2-1
24.6 19.7 20.0 22.8
0.10 ± 1.4 ± 0.9 ± 2.2 -+0.7
16.9 21.2 21.5 12.8
0.15 ± 0.5 -+0.6 +- 1.1 -+ 1.3
7.3 22.3 14.4 6.0
0.20 ± 0.8 +- 1.6 ± 1.1 ± 0.8
1.6 8.8 6.2 1.5
± 0.3 -+ 0.7 ± 0.5 ± 0.4
a Acid-soluble fragments from [methyl-3H] thymidine labelled M)NA at 30 °C expressed as percent of the amount released from denatured M)NA. Averages and standard errors of four determinations with two enzyme preparations Contributed by 0.01 M MgC12 (ionic strength 0.02), 0.01 M Tris-HC1 buffer pH 7.5 (ionic strength 0.01) and, the remainder, by KC1
chromatography the eluent was 0.I5 M potassium phosphate buffer pH 7.0, 0.25 M KC1, 1% Triton X-100. For hepaxinagarose chromatography the eluent was 0.35 M KC1, 0.01 M TrisHC1 buffer ph 7.5, 1% Triton X-100 (sic). Only the fractions with very high activity were pooled in order to avoid having to concentrate the sample. The sample was dialysed against 0.1 M KC1, 0.01 M Tris-HC1 and 1% Triton Xq00. With 1% Triton the enzyme solutions remained clear for several months in the refrigerator with no change in the activity. This endonuclease preparation is a stable complex, dissociated by SDS and resolved by polyacrylamide gel electrophoresis in four proteins (Morosoli and Lusena 1980).
Assays of endonucleases. The release of acid-soluble fragments from h-DNA labelled with (methyl-3H) thymine was measured by a method already described (Morosoli and Lusena 1982). The amount of nucleotides released was proportional to enzyme concentration and digestion time. Various ionic strengths were obtained by changing the KC1 concentrations. The endonuclease preparations were calibrated with denatured h-DNA at 0.08 ionic strength. In the range under study, ionic strength had no effect on the activity with denatured DNA. All strains yielded compa-
table amounts of endonuclease activity with denatured DNA. The results with native h-DNA were expressed as percent of the activity with denatured X-DNA and, therefore, when divided by 100, represent nmol of nucleotide released per min per enzyme unit (Morosoli and Lusena 1980). The release of acid soluble fragments from poly(dA)poly(dT) and poly(dA) • poly(dU) (P-L. Biochemicals) at 30 °C and 0.15 M ionic strength was measured spectrophotometrically (Morosoli and Lusena 1982). The endonuclease activity was expressed as percent of the activity with denatured X-DNA.
Results All three m u t a n t strains investigated had d e m o n s t r a t e d an increased sensitivity to petite-induction b y exposure to sulfanilamide-aminopterin with a c o n c o m i t a n t increase in petite p r o d u c t i o n after U V irradiation and after growth at an elevated t e m p e r a t u r e (Lusena and James 1982). For two o f these strains, # 2 2 and # 3 5 , genetic analyses d e m o n s t r a t e d that multiple sensitivity was attributable to n o n - c o m p l e m e n t i n g recessive m u t a t i o n s at a single locus (sasl-1 and sasl-2). For one strain # 3 4 , gegetic analyses d e m o n s t r a t e d the presence o f a recessive m u t a t i o n in another locus, sas2-1, conferring delayed sensitivity to sulfanilamide-aminopterin, U V sensitivity, b u t little or no sensitivity t o elevated t e m p e r a t u r e . Subsequent tests to compare endonuclease activities were carried out with segregants o f second-generation backcrosses o f the three strains genetically analysed ( # 2 2 , # 3 5 and # 3 4 ) . Table 1 presents data concerning the sensitivity o f these segregants to petite-inducing treatments. Multiple sensitivity has been retained b y # 2 2 (sasl-1)and b y # 3 5 (sasl-2),but # 3 4 (sas2-1)has lost its sensitivity to elevated t e m p e r a t u r e and shows sensitivity to drugs only after 72 h. Endonuclease activities were compared at four ionic strengths and the results are summarised in Table 2. The activity o f the wild t y p e e n z y m e decreased sharply w i t h increased ionic strength. In contrast the e n z y m e activities o f sasl-1 and sasl-2 were m u c h less affected b y changes in ionic strength. For the m u t a n t sas2-1 the enz y m e activity was not significantly different f r o m the wild-type. Clearly the t w o alMic m u t a t i o n s in the SAS1 gene resulted in an increased activity o f the m i t o c h o n d r i a l endonuclease with double stranded D N A at high ionic strength, i. e. w h e n the b o n d strength b e t w e e n strands was increased. M u t a t i o n in the SAS2 gene did n o t affect the e n z y m e activity. The abilities o f the wild-type and m u t a n t endonuclease to act on p o l y ( d A ) " p o l y ( d U ) and p o l y ( d A ) - p o l y ( d T ) , and to discriminate b e t w e e n p o l y ( d U ) and p o l y ( d T ) are shown in Table 3. The e n z y m e s isolated f r o m the sasl m u t a n t s have a m u c h higher activity w i t h b o t h substrates t h a n e n z y m e s f r o m wild t y p e cells. Their ability to discriminate b e t w e e n p o l y ( d T ) and p o l y ( d U ) is indicated
C. V. Lusena and C. Champagne: Yeast endonuclease involved in mt-DNA repair Table 3. Comparison of the endonuclease activitya of the mutants with polydA-polydU and polydA-polydT as substrate at 0.15 ionic strength Genotype
SAS sasl-1 sasl-2 sas2-1
Substrate
Ratio
AU
AT
16.2 36.4 21.9 10.5
3.4 t4.6 8.9 2.5
AU/AT 4.8 2.5 b 2.5 b 4.1
a Acid-soluble fragments released from synthetic polynucleotides at 30 °C expressed as percent of the amount released from denatured XDNA. Averages for two enzyme preparations b Values different from wild type at 1% level of significance
by the AU/AT ratio. These ratios show that the preparations from sasl mutants were not as discriminating as those from either the wild type or the sas2 mutant.
Discussion It is clear that the specificity o f the mitochondrial endonuctease is altered b y the presence o f either of two mutations, sasl-1, and sasl-2, in the SAS1 gene. These mutant enzyme activities are more active with doublestranded DNA than the enzyme from wild type cells when the hydrogen-bond strength between the strands is increased b y ionic strength or b y base composition. Therefore, the mutant enzyme activities are less able to discriminate between legitimate and mismatch base pairs. It is also clear that this loss of specificity is accompanied b y an increase in sensitivity of the mutant cells to induction o f petites b y exposure to sulfanilamide-aminopterin, ultraviolet irradiation, and elevated temperatures. All these treatments locally weaken the stability o f the double strand o f DNA. The mutant enzyme is not able to cope with these changes as efficiently as the normal enzyme. What is the role o f the mitochondrial endonuclease? We propose that the endonuclease under study in this report normally is responsible for the cuts necessary to eliminate mismatched regions in the mt-DNA. The normal enzyme in the wild-type strain can correct the errors induced b y the treatments described in this report, while the mutant enzyme cannot. After ultraviolet irradiation an extensive breakdown o f mt-DNA occurs (Moustacchi
21
et al. 1975) with selective degradation of A-T rich regions (Hixon and Moustacchi 1978). Similar degradation occurs after treatment with high concentrations o f sulfanilamide and aminopterin (Barclay and Little 1978). Both treatments yield petites. The remodelled DNA o f the resulting petites contains large deletions, rearrangements and repetitions. The endonuclease is responsible for the initial cuts, which are followed b y recombination and ligation (Gaillard et al. 1980). The role o f the endonuclease is therefore to perform the first step o f the repair mechanism o f mt-DNA and, when this repair is not performed adequately, it becomes part o f the process of petite production. Nucleases located in the nucleus and involved in the repair mechanisms for the removal o f mismatched segments are very specific (i. e. removal of thymidine dimers): a great deal of precision is required to protect the unique genome of each cell. Evidence for such enzymes has not been found in yeast mitochondria (Prakash 1975). Perhaps they are not necessary because there are many genomes in each cell. The mitochondrial endonuclease has a more general function: it preferentially cuts any region with lower attraction between the DNA strands. The process must be far from error-free, even under normal conditions, considering the high sensitivity o f yeast to petite induction. Acknowledgements. We thank Rosemary O'Connell for the data in Table 1 and Dr. A. P. James for discussions and criticisms of the manuscript.
References Barclay BJ, Little JG (1978) Mol Gen Genet 160:33-40 Gaillard C, Strauss F, Bernardi G (1980) Nature 283:218-220 Hixon S, Moustacchi E (1978) Biochem Biophys Res Commun 81:288-296 Heude M, Moustacchi E (1979) Genetics 93:81-103 Lusena CV, James AP (1982) Can J Genet Cyto124:781-786 Morosoli R, Lusena CV (1980) Eur J Biochem 110:431-437 Morosoli R, Lusena CV (1982) Can J Biochem 60:757-762 Moustacchi E, Waters R, Heude M, Chanet R (1975) In: Nygaard OF, Adler HI, Sinclair WK (eds) Radiation research, biomedical, chemical and physical perspectives. Academic Press, New York, pp 632 650 Prakash L (1975) J Mol Bio198:781-795 Schenberg-Frascino A, Moustacchi E (1972) Mol Gen Genet 115:243-257 Communicated b y R. J. Schweyen Received April 15 / July 4, 1983
