Current 6enetics
Current Genetics (1982) 6:29-30
© Springer-Verlag 1982
The Role of the cdc9 Ligase in Replication and Excision Repair in Saccharomyces cerevisiae S. J. McCready and B. S. Cox Botany School, South Parks Road, Oxford OX1 3RA, England
Summary. We show that the DNA ligase encoded or controlled by the cdc9 gene in Saccharomyces cerevisiae is required for replication ofplasmid DNA but that excision repair of pyrimidine dimers in plasmid DNA can be completed in its absence.
Materials and Methods Strains. The cdc9 strain used was cdc9-7 kindly provided by Dr
Lee Johnston. This allele is 'non-leaky' at the non-permissive temperature of 36 °C (Johnston, personal communication). Methods. Culture conditions, DNA extraction and the dimer
Key words: Yeast - Plasmid - Repair - Ligase
Introduction cdc9 mutants of the yeast, Saccharomyces cerevisiae, are unable to join Okazaki fragments at restrictive temperature and lack detectable DNA ligase activity at both restrictive (36 °C) and permissive (23 °C) temperatures (Johnston and Nasmyth 1978). This suggests that the cdc9 gene is either the structural gene for the major (or sole) DNA ligase in yeast or a gene controlling ligase levels. Johnston (1979) has shown that cdc9 mutants are more sensitive than wild-typecells to UV irradiation and suggested that the cdc9-dependent iigase is needed for the final step in excision repair. We have recently described an assay for pyrimidine dimers in yeast 2/.on circular DNA plasmid (McCready and Cox 1980). This plasmid has many features in common with chromosomal DNA and we have shown that the repair of both is dependent on most, probably all, of the same excision repair genes (McCready and Cox 1980 and unpublished data). We have now made use of this assay to determine whether the cdc9 ligase is needed a) for DNA replication in 2 pan plasmids and b) for excision repair of pyrimidine dimers from the plasmid DNA.
Offprint requests to: S. J. McCready
assay are as previouslydescribed except that, to test repair at 36 °C, cells were held in distilled water at 36 °C prior to irradiation and not exposed to a lower temperature at any stage before cell lysis and DNA extraction. DNA was extracted and exposed to Micrococcus luteus UV endonuclease to nick dimer-containing plasmids (McCready and Cox 1980). Agarose gel electrophoresis was used to separate nicked and convalently-closed.DNA molecules as described except that the electrophoresis buffer was 15 mM Tris, 18 mM NaH2PO4, 0.5 mM EDTA pH 7.8. The procedure for analysing gels was as described (McCready and Cox 1980).
Results and Discussion Separation of open circles (oc) and covalently closed circular (ccc) plasmid molecules on gels can be used to detect accumulation, during incubation, of nicked replication or repair intermediates. Separation of plasmids before and after UV-endonuclease treatment is used to measure the average number of dimers during incubation. The gel in Fig. 1 shows 1) that plasmids accumulate nicks in unirradiated cdc9-7 cells grown at restrictive temperature (tracks 1,4 and 7). After 2 h incubation at 36 °C abaout 50% ofplasmid molecules were in the oc form. This shows that the cdc9 ligase is used in plasmid DNA replication; 2) irradiated cells also accumulate open circles but more slowly (tracks 2, 5 and 8). Only 20% of plasmid molecules from irradiated cells have acquired nicks after 2 h. These nicked circles could be replication intermediates, the level of DNA synthesis having been de0172-8083/82/0006/0029/$ 01.00
30
S.J. McCready and B. S. Cox: Yeast edc9 Ligase in Plasmid Replication and Repair 60
"k\ \
~ \\
~oopen circles
\
\
\
~
\
\
110 UV
/ 111
I
~
0
incubation at 36 C (hours)
~ ~4~+ UV, +uv-erldO
I 2
Fig. IA. 1% agarose gel stained with ethidium bromide. Track 1, 4 and 7 show unirradiated DNA at time 0 and after 1 h and 2 h incubation at 37 °C in YEPD medium. Tracks 2, 5 and 8 show DNA from cells ffradiated at time 0 at 20 J. m -2, at time 0 and after 1 h and 2 h incubation at 37 °C in YEPD. Tracks 3, 6 and 9 are duplicates of samples 2, 5 and 8 incubated with M. luteus UV-endonuclease Fig. lB. Percentages of circles which are nicked, calculated from the gel shown in Fig 1A. - m - plasmids from unirradiated cells (from tracks 1, 4 and 7); - • - plasmids from cells irradiated at 20 J • m -2 (from tracks 2, 5 and 8); - + - plasmids from irradiated cells, exposed to UV-endonuclease (tracks 3, 6 and 9)
pressed by irradiation, or they could be repair intermediates (ff replication is completely stopped by irradiation at 20 J • m - 2 ) . We believe that they are more likely to be replication intermediates for two reasons: a) Wild-type cells are able to remove 90% of pyrimidine dimers from plasmid DNA during 2 h incubation (McCready and Cox 1980) after irradiation at 20 J . m - 2 . One would, therefore, expect that repair intermediates would be produced at this rate in a mutant unable to complete repair so that about 49% of plasmids would be in the oc form after 2 h post-UV incubation. b) Pyrimidine dirners in plasmids are being repaired. At time 0, 55% o f plasmid molecules contain one or m o r e pyrimidine dimers. After 2 h incubation, the UV-endonuclease treated sample contains only 25% open circles (track 9). Most of these are in the oc form even without UV-endonuclease treatment (track 8) and we do not know what proportion of them contain dimers. However, even if all of these were unrepaired or were repair intermediates, at least 55% of molecules have been completely repaired. If the 20% of plasmid molecules already in the oc form do not contain dimers we can conelude that 90% of dimers have been removed. We conclude that excision repair of dimers in 2 /am plasmid molecules can be completed in the absence of the cdc9 ligase.
A recent paper by Wilcox and Prakash (1981) shows that, in a cdc9 mutant, chromosomal DNA accumulates unrepaired nicks which do not appear in excision-defective mutants, suggesting that the cdc9 ligase is involved in excision repair of chromosomal DNA. Similar results have been obtained in our laboratory (J. Boyce, unpubfished). If these breaks are indeed a result of uncompleted excision repair, this suggests that the plasmid must have a ligase available to it which cannot be used for chromosomal DNA repair. A possible candidate for this function would be the plasmid-encoded FLP gene which promotes recombination between the inverted repeat sequences of 2 ~ plasmid molecules. Alternatively yeast may, in common with other eukaryotes, (Soderhall and Lindahl 1976) possess a second ligase. Acknowledgement. This work was funded by Science Research Council Grant no. GR/A 82253
References Johnston LH (1979) Mol Gen Genet 170 : 89-92 Johnston LH, Nasmyth KA (1978) Nature 274 : 891-893 McCready SJ, Cox BS (1980)Curt Genet 2:207-210 Soderhall S, Lindahl T (1976) FEBS Letters 67 : 1-8 Wilcox DR, Prakash L (1981) J Bacterio1148:618-623 Communicated by U. Leupold Received May 3/May 19, 1982
Current Genetics (1982) 6:29-30
© Springer-Verlag 1982
The Role of the cdc9 Ligase in Replication and Excision Repair in Saccharomyces cerevisiae S. J. McCready and B. S. Cox Botany School, South Parks Road, Oxford OX1 3RA, England
Summary. We show that the DNA ligase encoded or controlled by the cdc9 gene in Saccharomyces cerevisiae is required for replication ofplasmid DNA but that excision repair of pyrimidine dimers in plasmid DNA can be completed in its absence.
Materials and Methods Strains. The cdc9 strain used was cdc9-7 kindly provided by Dr
Lee Johnston. This allele is 'non-leaky' at the non-permissive temperature of 36 °C (Johnston, personal communication). Methods. Culture conditions, DNA extraction and the dimer
Key words: Yeast - Plasmid - Repair - Ligase
Introduction cdc9 mutants of the yeast, Saccharomyces cerevisiae, are unable to join Okazaki fragments at restrictive temperature and lack detectable DNA ligase activity at both restrictive (36 °C) and permissive (23 °C) temperatures (Johnston and Nasmyth 1978). This suggests that the cdc9 gene is either the structural gene for the major (or sole) DNA ligase in yeast or a gene controlling ligase levels. Johnston (1979) has shown that cdc9 mutants are more sensitive than wild-typecells to UV irradiation and suggested that the cdc9-dependent iigase is needed for the final step in excision repair. We have recently described an assay for pyrimidine dimers in yeast 2/.on circular DNA plasmid (McCready and Cox 1980). This plasmid has many features in common with chromosomal DNA and we have shown that the repair of both is dependent on most, probably all, of the same excision repair genes (McCready and Cox 1980 and unpublished data). We have now made use of this assay to determine whether the cdc9 ligase is needed a) for DNA replication in 2 pan plasmids and b) for excision repair of pyrimidine dimers from the plasmid DNA.
Offprint requests to: S. J. McCready
assay are as previouslydescribed except that, to test repair at 36 °C, cells were held in distilled water at 36 °C prior to irradiation and not exposed to a lower temperature at any stage before cell lysis and DNA extraction. DNA was extracted and exposed to Micrococcus luteus UV endonuclease to nick dimer-containing plasmids (McCready and Cox 1980). Agarose gel electrophoresis was used to separate nicked and convalently-closed.DNA molecules as described except that the electrophoresis buffer was 15 mM Tris, 18 mM NaH2PO4, 0.5 mM EDTA pH 7.8. The procedure for analysing gels was as described (McCready and Cox 1980).
Results and Discussion Separation of open circles (oc) and covalently closed circular (ccc) plasmid molecules on gels can be used to detect accumulation, during incubation, of nicked replication or repair intermediates. Separation of plasmids before and after UV-endonuclease treatment is used to measure the average number of dimers during incubation. The gel in Fig. 1 shows 1) that plasmids accumulate nicks in unirradiated cdc9-7 cells grown at restrictive temperature (tracks 1,4 and 7). After 2 h incubation at 36 °C abaout 50% ofplasmid molecules were in the oc form. This shows that the cdc9 ligase is used in plasmid DNA replication; 2) irradiated cells also accumulate open circles but more slowly (tracks 2, 5 and 8). Only 20% of plasmid molecules from irradiated cells have acquired nicks after 2 h. These nicked circles could be replication intermediates, the level of DNA synthesis having been de0172-8083/82/0006/0029/$ 01.00
30
S.J. McCready and B. S. Cox: Yeast edc9 Ligase in Plasmid Replication and Repair 60
"k\ \
~ \\
~oopen circles
\
\
\
~
\
\
110 UV
/ 111
I
~
0
incubation at 36 C (hours)
~ ~4~+ UV, +uv-erldO
I 2
Fig. IA. 1% agarose gel stained with ethidium bromide. Track 1, 4 and 7 show unirradiated DNA at time 0 and after 1 h and 2 h incubation at 37 °C in YEPD medium. Tracks 2, 5 and 8 show DNA from cells ffradiated at time 0 at 20 J. m -2, at time 0 and after 1 h and 2 h incubation at 37 °C in YEPD. Tracks 3, 6 and 9 are duplicates of samples 2, 5 and 8 incubated with M. luteus UV-endonuclease Fig. lB. Percentages of circles which are nicked, calculated from the gel shown in Fig 1A. - m - plasmids from unirradiated cells (from tracks 1, 4 and 7); - • - plasmids from cells irradiated at 20 J • m -2 (from tracks 2, 5 and 8); - + - plasmids from irradiated cells, exposed to UV-endonuclease (tracks 3, 6 and 9)
pressed by irradiation, or they could be repair intermediates (ff replication is completely stopped by irradiation at 20 J • m - 2 ) . We believe that they are more likely to be replication intermediates for two reasons: a) Wild-type cells are able to remove 90% of pyrimidine dimers from plasmid DNA during 2 h incubation (McCready and Cox 1980) after irradiation at 20 J . m - 2 . One would, therefore, expect that repair intermediates would be produced at this rate in a mutant unable to complete repair so that about 49% of plasmids would be in the oc form after 2 h post-UV incubation. b) Pyrimidine dirners in plasmids are being repaired. At time 0, 55% o f plasmid molecules contain one or m o r e pyrimidine dimers. After 2 h incubation, the UV-endonuclease treated sample contains only 25% open circles (track 9). Most of these are in the oc form even without UV-endonuclease treatment (track 8) and we do not know what proportion of them contain dimers. However, even if all of these were unrepaired or were repair intermediates, at least 55% of molecules have been completely repaired. If the 20% of plasmid molecules already in the oc form do not contain dimers we can conelude that 90% of dimers have been removed. We conclude that excision repair of dimers in 2 /am plasmid molecules can be completed in the absence of the cdc9 ligase.
A recent paper by Wilcox and Prakash (1981) shows that, in a cdc9 mutant, chromosomal DNA accumulates unrepaired nicks which do not appear in excision-defective mutants, suggesting that the cdc9 ligase is involved in excision repair of chromosomal DNA. Similar results have been obtained in our laboratory (J. Boyce, unpubfished). If these breaks are indeed a result of uncompleted excision repair, this suggests that the plasmid must have a ligase available to it which cannot be used for chromosomal DNA repair. A possible candidate for this function would be the plasmid-encoded FLP gene which promotes recombination between the inverted repeat sequences of 2 ~ plasmid molecules. Alternatively yeast may, in common with other eukaryotes, (Soderhall and Lindahl 1976) possess a second ligase. Acknowledgement. This work was funded by Science Research Council Grant no. GR/A 82253
References Johnston LH (1979) Mol Gen Genet 170 : 89-92 Johnston LH, Nasmyth KA (1978) Nature 274 : 891-893 McCready SJ, Cox BS (1980)Curt Genet 2:207-210 Soderhall S, Lindahl T (1976) FEBS Letters 67 : 1-8 Wilcox DR, Prakash L (1981) J Bacterio1148:618-623 Communicated by U. Leupold Received May 3/May 19, 1982
