Mol Gen Genet (1985) 201:99-106 © Springer-Verlag1985

The use of plasmid D N A to probe D N A repair functions in the yeast Saccharomyces cerevisiae Charles I. White and Steven G. Sedgwick Genetics Division, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, U.K.

Summary. The survival of plasmid Y R p l 2 treated in vitro with ultraviolet- or 7-radiation, or with restriction endonucleases, has been used to investigate in vivo RAD gene activity in Saccharomyces cerevisiae. Yields of pyrmidine dimers or single and double strand breaks in plasmid D N A were assayed by physical methods. The biological effects of these damages were assayed by transformation of wildtype cells and rad mutants from each of the major groups of radiosensitive mutants. After UV-irradiation plasmid survival depended qualitatively on the same host functions that are needed for cellular survival. After 7-irradiation no such correspondence was found. Apart from a RAD52-dependent stimulation of transformation efficiency at low doses, other host repair functions had little effect. Stimulation of transformation corresponded with the production of double- but not single-strand breaks in plasmid sequences homologous with the yeast genome and may be linked with a transient increase in mitotic stability. More generally these data also show that transformation events using the LiC1 protocol may entail the uptake of a very low number of plasmid molecules per cell over a 10-fold range of D N A concentrations.

Introduction

Three D N A repair pathways have been defined in Saccharomyces cerevisiae on the basis of epistatic interactions between mutations conferring radiation sensitivity (Cox and Game 1974). The three classes of mutations correspond to three groups defined by differing relative sensitivities to ionizing and non-ionizing radiations. One class, sensitive to ultraviolet light (UV) but not ionizing radiation, lacks an excision repair system, radl, rad2, rad3, rad4 and radlO mutants belong to this epistasis group and are deficient in making the initial damage-specific endonucleolytic incision (Prakash 1977a, b; Reynolds and Friedberg 1981; Wilcox and Prakash 1981). Mutants such as rad7, radl4 and rad16 show only partial and less well understood deficiencies in excision repair. A second radiation repair pathway is concerned primarily with damage produced by ionizing radiation. The properties of mutants in this pathway show some heterogeneity. rad51, rad52 and rad54 cells are most sensitive, they are defective in both induced and spontaneous mitotic recombi-

Offprint requests to: C.I. White

nation (Saeki et al. 1980), and rad52 and rad54-3 mutants are unable to repair double-strand D N A breaks induced by ionizing radiation (Ho 1975; Resnick and Martin 1976; Budd and Mortimer 1982). Other mutants in this epistasis group, such as rad50, tad53, rad55, rad56 and rad57, are less radiosensitive. They have some recombination ability and one, tad50, is known to repair double-strand breaks (reviewed by Game 1982). RAD5, RAD6, RAD8, RAD9, RAD15 and RAD18 genes are needed for the third D N A repair pathway, which confers resistance to both UV and ionizing radiations. This so-called RAD6 epistasis group includes all known mutants deficient in induced mutagenesis, though not all mutations within the group, for example rad9-1, share this phenotype. With the exception of rad6 and radl8, mutants in this group show less UV sensitivity than excision defective strains. rad6-1, rad9-1 and radl8-1, have been shown to be competent in excision repair (Prakash 1977 a; Reynolds and Friedberg 1981). The complex and heterogeneous phenotypes of mutants within this group argue for the existence of both non-mutagenic and mutagenic repair processes under RAD6 control. The properties of these mutants are discussed in a number of recent reviews (Haynes and Kunz 1981 ; Lawrence 1982; Game 1982). One strategy for further studies of RAD gene activity, explored in the work described here, depends on the use of irradiated plasmids as substrates for the assay in vivo D N A repair. Plasmid D N A has the advantage of being relatively small, with known nucleotide sequence. It can be manipulated to produce defined amounts of D N A damage which can be assessed for effects on survival, mutagenesis and recombination. As a first step in this approach, the survival of UV and 7-irradiated plasmid D N A was determined after transformation of wild-type yeast and a representative rad mutant from each epistasis group. Materials and methods

Media The complete yeast extract-peptone-glucose, (YEPD) and synthetic minimal media, and supplements are as described in Sherman et al. (1971).

Yeast. Listed in Table 1 are the rad mutants used in this work. Strains C3-7, C4-5, C5-6, C6-3 and C8-2 are progeny of matings between YNN27 (RAD, from Dr. G. Banks) and mutants kindly sent by J. Game, J. Boyce and B. Cox;

100 Table 1. Yeast strains used in this work Strain

Relevant genotype

YNN27 C4-5 DBY747 + LP9 LP2649-1tc LP2693-4b C6-3 C3-7 C8-2 C5-6

RAD trpl-289-ura3-52 radl-1 trpl-289 ura3-52 rad2A : : URA3-trpl-289, ura3-52 rad3-2 ura3-5 2 rad4-4 trp l-289 ura3-52 rad6-1 trpl-289 ura3-52 rad7-1 trp1-289 ura3-52 radl4 ura3-52 rad52-1 trp1-289 ura3-52

For the origin of strains see Materials and methods

others are from S. Prakash. The radl4 allele number is unknown but is the original isolate from B. Cox. Matings were carried out using standard techniques (Sherman et al. 1971); random spores were screened for radiosensitivity, purified as single colonies, rescreened for radiation sensitivity and their auxotrophic phenotypes determined. Plasmid D N A

The yeast E. coli shuttle vector Y R p l 2 has approximately 6900 bp and consists of pBR322, with the yeast A R S 1 T R P ! and U R A 3 genes inserted into the E c o R I and AvaI sites respectively (Scherer and Davis 1979). Plasmid YCp50 has 8040 bp and contains the centromeric D N A of yeast chromosome 4(CEN4), A R S 1 and U R A 3 genes (from C. Mann). Plasmid pMA9 has approximately 7000 bp, including the centromeric D N A of yeast chromosome 3(CEN3) and the A R S 1 - T R P 1 genes (Clarke and Carbon 1980). Plasmid D N A was prepared from recA56 E. coli, by the alkaline lysis procedure and purified by equilibrium density gradient centrifugation in CsC1 (Maniatis et al. 1982).

mined with an 'UVX-Radiometer' (Ultraviolet Products Ltd., Cambridge, England). Fifty microliter aliquots of D N A were y-irradiated with a 'Gammabeam 650' 6°Co source at a rate of 26 Krads min- 1 Physical measurement o f damage to D N A UV. A saturating amount of crude M . luteus UV endonuclease was incubated with 2 lag samples of irradiated plasmid D N A in 10 mM Tris-HC1, pH 7.4, 50 mM NaC1, 1 mM NazEDTA at 37 ° C for 30 min (Haseltine et al. 1980). The samples were electrophoresed in an 0.7% agarose gel, stained with EtBr and photographed. The photograph was scanned with a Joyce Loebl 'Chromoscan 3' densitometer. The Poisson distribution was used to quantitate the production of enzyme-sensitive sites, or pyrimidine dimers, from the relative decrease in the amount of D N A present in the supercoiled form. All samples had the same exposure to the UV endonuclease and thus non-specific activity would not affect the observed relationship between UV dose and loss of supercoiled molecules. The D N A topoisomer UV lesion assay was described by Ciarrocchi and Pedrini (1982), who used it to determine the D N A helix unwinding angle of a pyrimidine dimer induced by ultraviolet radiation. Plasmid D N A was partially relaxed with D N A topoisomerase I from Ustilago maydis (a gift from Dr. A. Morrison) in the presence of 6 lag/ml ethidium bromide. This material migrates in an agarose gel as a ladder of bands representing a population of molecules with different, integral numbers of superhelical turns. After phenol/CHC13 and ether extraction, samples were UV-irradiated and electrophoresed through an 0.7% agarose gel, which was then photographed and scanned. The position of the peak absorbance of each band was expressed relative to the front of the fully supercoiled D N A band to measure the unwinding of supercoiled plasmid D N A by UV.

Transformation Bacteria. E. coli cells were transformed by the CaC12 procedure (Mandel and Higa 1970) with minor modifications. Yeast. Yeast cells were transformed by the LiC1 method (Ito et al. 1983). Each transformation consisted of approximately 108 cells, 3-4 lag plasmid DNA, 20 lag denatured salmon sperm DNA, and was plated directly on yeast minimal medium with the necessary supplements. The concentration of plasmid D N A used was determined to be nonsaturating in terms of transformants per lag D N A for each of the strains used. Manipulations were carried out in semidark conditions to minimize photoreactivation. Colonies transformed to U R A ÷ TRP ÷ were scored after 4-5 days growth at 32 ° C. Control transformations without plasmid D N A showed no observable spontaneous reversion of selected phenotype. Irradiation o f D N A

Plasmid D N A was irradiated at an approximate concentration of 250 lag/ml in 10 mM Tris-HC1, pH 7.4, 2.5 mM Na2EDTA. For UV-irradiation, 15-30 lal droplets were exposed on the surface of a plastic Petri dish to a Hanovia low pressure germicidal lamp at an incident flux of 3.35 Jm-2s-1. Before each experiment the lamp flux was deter-

)~-rays. Aliquots of y-irradiated D N A were electrophoresed in an 0.7% agarose gel, the gel photographed and scanned as above to determine relative amounts of supercoiled, relaxed circular and linear molecules. To allow for the effect of D N A topology on the extent of ethidium bromide intercalation, the relative fluorescence of the supercoiled form was multiplied by a factor of 1.36 (Projan et al. 1983). Production and assay o f nicked and linearized plasmid D N A

Restriction endonucleases B a m H I and XbaI (Bethesda Research Labs., Maryland, USA), were used under standard conditions (Maniatis et al. 1982) to produce linear plasmid D N A cut specifically within yeast (XbaI) or vector (BamHI) sequences. Digestions were also carried out in the presence of ethidium bromide (20 and 200 gg/ml), in order to produce relaxed circular molecules cut specifically in one D N A strand only ((}sterlund et al. 1982). After enzymatic treatment, the D N A was phenol and chloroform extracted followed by ethanol precipitation. D N A pellets were dried, redissolved in 10 mM Tris-HC1, pH 7.4, 2.5 mM EDTA, and samples of each were electrophoresed in a slab agarose gel. A photograph of the gel was scanned to measure the proportions of the supercoiled, nicked-circular and linear forms of the plasmid.

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Results

Physical assays of plasmid damage UV. The incidence o f UV p h o t o p r o d u c t s in Y R p l 2 D N A was quantitated in two ways. This was necessary as the UV-endonuclease assay is useful only at lower doses than those used for the plasmid survival experiments. UV-endonucleolytic conversion o f lightly U V - i r r a d i a t e d supercoiled D N A to open circles showed the production o f a mean o f one U V endonuclease sensitive site, or pyrimidine dimer, per molecule per 21 Jm 2 (Fig. i a). This is equivalent to 1.04 dimers per 108 daltons J - t i n 2, which compares favourably with values o f 1.43 and 2.09 dimers per 10 s daltons J - ~ m - 2 determined chromatographically for E. coli (Sedgwick 1975) and ColE1 D N A (Seawell et al. 1980) respectively. A t higher doses the a m o u n t of p h o t o p r o d u c t s continued to increase linearly to 350 Jm -2 as judged by the progressive loss o f superhelicity with increasing UV dose (Fig. l b ) . The d a t a o f W a c k e r et al. (1960), indicate that this linear relationship holds up to a dose o f at least 3,600 Jm -2, which is greater than the highest used in our w o r k (2,680 J m - 2). These combined d a t a therefore indicate that one superhelical turn was lost per 650 Jm -2 or per 31 pyrimidine dimers. This corresponds to - 12 ° of unwinding per pyridine dimer and is in g o o d agreement with the value o f - 14.3 ° obtained by Ciarrocchi and Pedrini (1982). y-rays. The rate of loss of plasmid D N A from the supercoiled form corresponds to the mean production o f one single-strand break per YRp12 molecule per 15 K r a d s (Fig. 2a). There was no observable supercoiled D N A at higher doses and the loss o f D N A from the nicked circular form was used to quantitate the induction o f double-strand breaks. A p p r o x i m a t e l y 200 K r a d s was found to induce a mean o f one double-strand break per molecule. This is equivalent to 1.02-10-9 double strand b r e a k s / d a l t o n / K r a d and similar to, buit higher than, values of 0 . 4 - 2 . 1 0 - t ° / d a l t o n / K r a d reported by a number o f authors for bacterial,

Fig. 2a, b. in vitro assay of D N A strand breakage in (a), and transformation of (b) y-irradiated DNA. a Relative amounts o£ supercoiled (i), relaxed-circular (o) and linear (,) forms of YRp12 D N A given a range of y-ray doses (see Methods). b Transformation

efficiencies of y-irradiated, relative to unirradiated, YRpI2 DNA in RAD (o), rad6-1 (zx), tad1-1 (u) and rad52-1 (v) yeast cells

yeast and m a m m a l i a n cells (from Resnick and Martin 1976). The absence o f proteins etc. which make up the intranuclear environment might be expected to increase the susceptibility of D N A to y-rays. These breaks consist o f double strand breaks and pairs of single strand breaks sufficiently close on opposite strands that the intervening D N A helix is unstable. The n u m b e r o f the latter were calculated from the single strand breakage yield per molecule, S, and the equation: D = (2b + 1) (S/M) 2 .M/2 where D is the double strand break yield, M the number o f bases per molecule and b the m a x i m u m distance a p a r t in bases between the two single strand breaks (Schumaker et al. 1956). W h e n b is 6-12 bases, the p r o p o r t i o n of double strand breaks p r o d u c e d by close single strand breaks was calculated to be 0.08-0.16 per molecule. As a p r o p o r t i o n o f the molecules were in the open circular form at zero dose, this figure is a slight underestimate of the actual value.

Irradiated plasmid DNA and transformation The survival o f p l a s m i d s treated with UV, y-rays or endonucleases was determined after transformation o f wild-type and D N A repair deficient yeast. Survival was calculated as the number o f transformants at each dose relative to the number o f transformants at the zero dose.

UV-irradiatedplasmid DNA. The survival o f UV-irradiated YRp12 plasmid D N A in wild-type, RAD, yeast showed a shoulder up to 650 Jm - z followed by an exponential de-

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Fig. 3. Transforming efficiencies of UVirradiated, relative to unirradiated, YRp12 DNA in: a) excision defective radl-1 (,.), rad2A (t), rad3-2 (A), rad4-4 (,), tad7-1 (.) and radl4 (0) cells, b RAD (o), tad52-1 (v), and rad6-1 (z~) yeast cells

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crease (Fig. 3b). The slope of this region corresponds to the incidence of one lethal event per 495 Jm 2, or per 23.5 pyrimidine dimers. The rad52-1 mutant showed a similar relationship between plasmid survival and UV dose to the wild-type (RAD), though with a less pronounced shoulder at low doses. In the presence of the radl group mutations, radl-1, rad2A, rad3-2, rad4-4, rad7-1 and rad14, the survival of UV-irradiated plasmid was markedly different from that in the RAD, wild-type cells (Fig. 3 a). N o shoulder at low doses was observed and plasmid survival decreased exponentially with increasing dose. Plasmid survival decreased at a rate corresponding to the incidence of one lethal hit per 50.25 J m - 2 or per 2.4 pyrimidine dimers per plasmid molecule in the most sensitive radl-1 and rad3-2 mutants. Transformation of the rad6-1 mutant resulted in a plasmid survival curve intermediate between the wild-type (and rad52-1) and excision repair defective (radl-1) mutants. In rad6-1 cells the mean dose for one lethal event per plasmid was 314 Jm -2, which corresponds to a yield of 15 pyrimidine dimers per plasmid. Similar decreases in the survival of UV-irradiated plasmid were seen across a 10-fold range of D N A concentrations in R A D + cells (Fig. 4a). This indicates that these assays o f plasmid survival are not influenced by multiplicity effects which could mask lethal effects by increasing the number of plasmid molecules either entering or becoming established in each cell, and perhaps permitting multiplicity reactivation by inter-plasmid recombination. In radl-1 cells there was greater apparent inactivation of plasmid at high concentrations of transforming D N A (Fig. 4c). Although the cause of this is unknown, it does show that for radl-1, as for R A D cells, the multiplicity effects described above do not influence the plasmid survival assays.

7-irradiated plasmid DNA. All mutants tested, except rad52-1, resembled the wild-type in their ability to be transformed with 7-irradiated YRp12 D N A (Fig. 2b). The RAD52 cells displayed a 1.4 to 2.5 fold increase in transfor-

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Fig. 4a-ft. Transformation of wild-type (a, b), radi-i (e) and rad52-1 (fl) yeast cells with increasing amounts of unirradiated (m), UV- (A) or 7-irradiated (i) YRp12 DNA. Plasmid DNA was irradiated with: 1,700 Jm -2 (a) or 185 Jm -2 (e) UV light, or 50 Krads 7-rays (b, d)

mation efficiency at low doses of 7-rays to the plasmid, followed by an exponential decay in plasmid survival corresponding to approximately one lethal hit per 3(~40 Krads. The survival of 7-irradiated plasmid in the rad52-1 mutant yeast show an exponential decrease, corresponding to one lethal hit per 20 Krads, with no observable shoulder. The lesion and effects stimulating transformation efficiency were further investigated. Firstly, the relative effects of double and single-strand breakage were assessed by mak-

103 Table 2. Transformation of RAD and rad52-1 cells under standardized conditions with untreated and endonuclease digested YRpl2 DNA. Both enzymes cleave the plasmid only once, BamHI in the pBR322 sequence and Xbal in the yeast sequence. Numbers given are transformants per plate

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Fig. 5. Transformation of strain C4-5 (radl-1) with increasing proportion of XbaI linearized vs. circular YRpl2 DNA: unirradiated DNA (n); UV-irradiated DNA (185 Jm -z) (m)

ing cuts in plasmid D N A with restriction endonucleases. Linearization of plasmid D N A by cutting within the yeast sequence with XbaI, stimulated the transformation of R A D cells 17-fold relative to untreated D N A but caused a 2-3-fold decrease in transformation in rad52-1 mutants. In R A D cells cutting in the pBR322-derived sequence with BamHI doubled the transformation efficiency while in rad52-1 mutants transformation efficiency was reduced 16-fold (Table 2). It should be noted that some variation in the magnitudes of these effects was seen between different preparations of competent cells (see, for example, Fig. 5). In contrast, single-strand nicking of plasmid D N A had a much smaller effect on transformation efficiency. Treatment of YRpl 2 D N A containing 4% presumably randomly nicked molecules with XbaI to give 16% nicked molecules caused no equivalent increase in transformation efficiency. In fact the slightly enhanced transforming ability of this material can be ascribed to the small increase in linear DNA. D N A obtained under conditions producing fewer nicked circles and more linear molecules transformed more

Fig. 6. Transformation efficiencies of y-irradiated centromeric plasraids in wild-type cells: YCp50 (o); pMA9 (e)

efficiently; thus, conversion of nicked to linear molecules stimulated transformation. Similar experiments with BarnHI nicked D N A showed no stimulatory effect of single strand breakage in pBR322-derived sequences on transformation efficiency (Table 2). Variations in the D N A concentration did not alter the extent of the stimulatory effect of low 7-ray dose upon transformation of R A D 5 2 cells, nor did they affect the amount of sensitization in rad52 mutants (Fig. 4b, d). Furthermore no change in the extent of killing of UV-irradiated plasmid were seen with increasing relative concentrations of linear molecules (Fig. 5). Thus, the transformation with y-irradiated plasmid, as with UV-irradiated DNA, shows an absence of multiplicity effects due to variations in the amounts of D N A or proportion of linearized molecules. A second explanation for the stimulation of transformation efficiency is that it involves a homology-dependent interaction between plasmid and yeast sequences. With nonreplicative plasmids such an event is well documented and results in the integration of the plasmid into the yeast genome (Orr-Weaver et al. 1981). However, cells transformed with either supercoiled or linear YRp12 displayed the same mitotic instability as judged by the loss of transformed phenotype during non-selective growth (data not shown). This indicates that linear plasmids had not been stably integrated into the yeast genome although it is possible that transient integration does occur (see Discussion). Similarly, Southern hybridization did not reveal the presence of integrated YRp12 sequences in genomic D N A (data not shown). A third possibility was that double-strand breakage somehow increased the likelihood of a transformant becoming established once the plasmid D N A has entered the cell. This possibility is supported by the observation that neither low 7-ray doses (Fig. 6), nor linearization within yeast D N A (data not shown) stimulated the transforming ability of the centromeric plasmids YCp50 and pMA9. The same preparations of cells did show increased transformation with both endonuclease and ?,-ray treated YRp12 D N A (data not shown). These C E N plasmids are already mitotically stable, in marked contrast to YRp12 (Stinchcomb etal. 1979; Clarke and Carbon 1980; Murray and Szostak 1983). The absence of the stimulatory effects on them therefore indicates that the increased transforming ability of linear YRp12 molecules is due to stability effects. Discussion

We have used plasmid D N A as substrate for assays of in vivo D N A repair activity in S. cerevisiae. Small plasmid

104 size permits defined D N A lesions and alterations to be made and assayed for biological effect. A potential problem was that the physical changes wrought by UV and 7-irradiation might produce molecules with different uptake characteristics compared to the untreated plasmid. Transformation efficiency responded similarly to increasing concentrations of untreated, UV or y-irradiated D N A suggesting that D N A uptake is not grossly affected by the doses given in this work. Furthermore these data strongly indicate that a very low number of plasmid molecules is introduced into each competent cell and that this number is largely independent of D N A concentration. Had this not been so the lethal effects of irradiation would have been less apparent at higher D N A concentrations owing to the greater probability of transformation with more than one molecule. The absence of shoulders on the survival curves also indicates that a low number of plasmids enter each competent cell. The possibility that a greater number of plasmids enter each competent cell and that one of these is chosen at random for replication cannot be excluded by our data, but is unlikely for the following reasons. Firstly, Y R p l 2 exists at an estimated 38-92 copies per transformed cell (Hyman et al. 1982) and secondly at least 95% of the A R S plasmids in a population of transformed cells are replicated once per cell cycle (Fangman et al. 1983). Thus it seems very unlikely that any particular cell is able to establish only one molecule of those that enter the cell. With UV radiation, the survival of the irradiated plasmid D N A qualitatively reflects the cellular radioresistance. Excision repair deficient radl group mutants show the greatest sensitivity of both cellular and plasmid viability (Fig. 3) to UV radiation. Similarly, the tad52-1 mutants, which have only slight cellular radiosensitivity, showed approximately wild-type levels of UV-irradiated plasmid survival. An exception arose with rad6-1 mutants where survival of UV-irradiated plasmid was intermediate between that seen in the wild-type and excision defective radl-1 mutants, though rad6-1 cells exhibited extreme UV-sensitivity. Thus, in this mutant, the survival of UV-irradiated plasmid does not directly reflect the cellular radiosensitivity. Possible reasons for this may include differences in photoproduct yield, type and repairability in chromatin and naked DNA. In addition some transformants may have suffered deletions in non-essential regions of the plasmid which in chromosomal D N A could have lethal effect. Such possibilities may readily be further investigated using plasmid assays. In contrast to the above results with excision deficient mutants, a recent paper (Dominski and Jachymczyk 1984) reported no significant difference between the survival of UV-irradiated plasmid D N A in wild-type and rad3-2 cells. Although a different plasmid was used, based on the 2 gm replicon, its radiosensitivity was similar to that presented here for RAD cells. This argues against major differences in transformation protocols causing the lack of sensitivity in the rad3 cells. It is noted that our rad3-2 strain, in agreement with the data of Prakash (1977 a), shows approximately 10-fold greater cellular UV-sensitivity than that reported by Dominski and Jachymczyk (1984). Using another rad3-2 strain (CM-3d, from J. Boyce) we have found a plasmid UV-survival response identical to that shown in Fig. 2a for rad3-2 cells. Possibly the cells of Dominski and Jachymczyk have accumulated suppressors of the rad3-2 allele which may explain the difference between our observations. The transformation efficiency of 7-irradiated plasmid

D N A does not reflect the cellular sensitivities of the host cells used. With the exception of rad52-1, all cells, whether radiation resistant or sensitive, showed a similar effect of ?-rays on plasmid survival. 7-rays appear to have two opposing effects on the transforming ability of plasmid DNA. At low ?-ray doses a stimulatory effect dependent on RAD52 gene function counters plasmid inactivation which becomes more apparent at higher doses. The exponential killing component of these survival curves was similar in both RAD and rad52-1 cells, but did not correspond with either double- or single-strand breakage frequencies. Endonucleolytic linearization of the plasmid showed that the stimulation could be due to double-strand breakage within plasmid D N A homologous to yeast chromosomal sequences. As linearization within plasmid-specific sequences gave no transformation stimulation, the effect is not due to plasmid-plasmid interactions within the cell. The likelihood of these interactions would be further reduced if, as argued, a low number of plasmids enters each cell. Either increased uptake or increased stability of linear relative to circular molecules could cause this linearizationdependent stimulation of transforming ability. Two distinct mechanisms for the increased uptake of linear molecules may be envisaged: either an increased • number of molecules are taken up by each competent cell or a greater number of cells are competent to take up linear DNA. If more linear molecules were taken up per competent cell, then UV-irradiated linear plasmid D N A would be expected to have greater survival than UV-irradiated circular molecules. Increased multiplicity would reduce the likelihood of all incoming molecules being inactivated and could increase the chance of recombinational reactivation. However linearized D N A showed the same extent of UV killing as circular plasmid (Fig. 5). Thus linearization does not appear to be increasing the number of molecules entering each cell. The other possibility is that a greater number of cells in the population are capable of taking up linearized D N A and that the difference between plasmid cut within yeast or vector D N A might reflect a mechanism in RAD52 cells which heals broken molecules in a chromosomal-homology dependent manner. Although this mechanism cannot be entirely discounted, it appears unlikely on two counts. Firstly centromeric plasmid D N A showed the same transforming ability whether linearized or not (data not shown). Secondly the relationship between D N A concentration and transformation efficiency was similar for linear and supercoiled D N A (Fig. 4). Thus neither scheme for increased uptake of linear D N A is applicable. Rather the results point to linearization increasing plasmid stability. A R S containing plasmids such as YRp12 are mitotically unstable in yeast (see Murray and Szostak 1983) and it is probable, if one or very few plasmids enter each competent cell, that many cells which take up plasmid D N A do not survive to form transformant colonies. Thus it is possible that by increasing the mitotic stability of the plasmid, even for a few divisions, linearization could boost the number of transformant colonies obtained. Linearization of non-replicative plasmids stimulates their integration into the yeast chromosome by homologydependent recombination between the breakpoint and integration site (Orr-Weaver et al. 1981). These integrated plasraids are mitotically stable. However, mitotic stability tests and Southern hybridization showed that linearization of the replicative plasmid YRp12 did not cause long term sta-

105 bilization through integrative recombination (data not shown). If integration does occur, excision of the integrated D N A must happen very early in the life of the transformant colony due to the poor growth under selection of episomal A R S plasmid transformants relative to the mitotically stable integrant transformants. Cells within an integrant colony which suffered a n excision of the integrated plasmid would be overgrown by their more stable sisters and the colony would retain the characteristic stable phenotype of the integrant transformants (see M u r r a y and Szostak 1983). Nevertheless, the possibility that linearization caused a short term increase in stability is strongly indicated by the lack of stimulation of transformation with linearized centromeric plasraids. As these plasmids are already mitotically stable in yeast cells, linearization would not be expected to further stimulate their transforming ability. Thus we suggest the stimulated transforming ability of linearized Y R p l 2 D N A may be caused by a homology-dependent interaction with the chromosomal D N A giving transiently enhanced stability. Although the stimulation of chromosomal interaction was absent from rad52-1 cells, there were some indications of low levels of recombination in these mutants. The plasmid cut within the yeast D N A transformed rad52-1 cells more efficiently than that cut within the vector DNA. Thus plasmid-chromosome interactions may have some effect on transformation even in the absence of the R A D 5 2 gene product. The reduction but not elimination of RAD52-dependent events in rad52-1 cells has been described for linear plasmid integration (Orr-Weaver et al. 1981), conversion between chromosomal duplications (Jackson and Fink 1981), and inter-plasmid recombination (Symington et al. 1983; Whiteway and A h m e d 1983). This effect may be due to leakiness of the rad52-1 allele or the presence of an alternate pathway, or pathways, through which these events may be mediated. These experiments show that certain in vivo D N A repair activities can be studied with plasmid systems. Although plasmid-specific effects, such as the stimulation of transforming ability of YRp12 D N A at low y-ray doses, can interfere, they may be identified and quantitated. Acknowledgements. We thank Drs. J. Game, J. Boyce, B. Cox, S. Prakash and C. Mann for their gifts of strains and plasmids. Also A. Morrison, R. Holliday, G. Banks and A. Spanos are thanked for helpful discussion and comments.

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