Proc. Natl. Acad. Sci. USA Vol. 81, pp. 7156-7160, November 1984 mitochondrial genomes consisting of only AT base pairs replicate and exhibit suppressiveness (/replication origins/DNA excision) WALTON L. FANGMAN* AND BERNARD DUJON Centre de Gdndtique Moldculaire, Centre National de la Recherche Scientifique, 91190 Gif-sur-Yvette, France Communicated by Herschel L. Roman, July 16, 1984

ABSTRACT Mutants, called p-, that result from exten- genome in the zygote. The p- genome, in a fraction of the sive deletions of the 75-kilobase Saccharomyces cerevisiae zygotes, monopolizes the mitochondrial replication or segre- mitochondrial genome arise at high frequency. The remaining gation apparatus, excluding the p+ genome, which is ulti- mitochondrial DNA is amplified in the p- cells, often as head- mately destroyed or diluted out. [Recombination between to-tail multimers, producing a cell with the normal amount of the p- and the p+ genomes resulting in destruction of respi- mitochondrial DNA. In matings, some of these p- mutants ex- ratory competence has also been postulated as a mechanism hibit zygotic hypersuppressiveness, excluding the wild-type for suppressiveness. This mechanism is ruled out because mitochondrial genome (p+) from all the diploids that are pro- the p- mitochondrial DNA of the zygotic progeny is the duced. From a hypersuppressive p- strain, we isolated two same as the p- parent (7, 8).] p- mutants that exhibit >95% mutants with reduced suppressiveness. These mutants, one zygotic suppressiveness have been called hypersuppressives moderately suppressive and one nonsuppressive, retain only (9). Hypersuppressive p- mutants retain a short [usually 89 and 70 base pairs, respectively, of the wild-type mitochon- s2500 base pairs (bp)] tandemly repeated segment that drial genome. Their sequences consist of 100% A-T base arises from only three different regions of the p+ genome of pairs. Replication of DNA in the , formation the strain KL14-4A. Each hypersuppressive p- contains a and amplification of new deletion genomes, and exhibition of 300-bp stretch that is almost identical (8-11). These 300-bp suppressiveness do not require a single G C base pair. sequences in the three different regions have been called rep1, rep2, and rep3. While the rep sequences may corre- The dispensable of the Saccharomyces cerevisiae spond to three major origins of replication used by the p+ mitochondrial genome has allowed an extensive genetic genome, a role in another process, such as mitochondrial analysis of mitochondrial DNA gene organization and genome segregation, has not been excluded. expression (reviewed in ref. 1). However, because the mito- The p- genome HS3324, which contains rep2, exhibits au- chondrial genome cannot be isolated as a discrete DNA mol- tonomous replication sequence (ARS) activity when trans- ecule but only as fragments, the molecular details of its repli- formed into yeast cells as part of an -yeast cation have not been determined. The wild-type 75-kilobase chimeric (8). The transformed plasmid appears to (kb) circular mitochondrial genome (p+) gives rise, by mas- reside in the yeast nucleus. Deletion analysis of the cloned sive deletion of p+ DNA, to respiratory-deficient mutants, 963-bp HS3324 DNA has shown that ARS activity requires p-s, at a high frequency (reviewed in ref. 1). Two observa- both a G+C-rich segment (Fig. 1, I) in the rep2 sequence, tions about p- mutants are clearly related to the mechanism and a short sequence from one of two A+T-rich segments (B of mitochondrial DNA replication. First, p- strains contain or B') in flanking unique DNA (11). Thus, while hyper- the same amount of mitochondrial DNA per cell as does the suppressiveness in the mitochondria may require only rep p+ wild type. That is, the DNA segment retained in a dele- sequences, ARS activity in the nucleus requires a second tion mutant is amplified during or immediately after the dele- element. tion event, and it is often present as a series of head-to-tail We began an analysis to determine more directly which circular multimers (although inverted repeats and more com- particular sequences in the mitochondrial DNA of a hyper- plex arrangements also exist). Second, p- mutants can arise suppressive p- mutant are required for suppressiveness in from many different regions of the p+ genome. Therefore, vivo. Using the HS3324 hypersuppressive p- strain, we iso- many different segments of the p+ genome are capable of lated mutants with reduced suppressiveness. The rationale promoting their own replication in the mitochondrion. was to determine whether such mutants had altered or de- When two genetically marked p+ strains are mated, the leted sequences in the rep region, the secondary elements resulting diploid segregates the different parental and recom- required for ARS activity, or other regions of the hyper- binant p+ genomes within a few cell divisions (2-4). Matings suppressive p- DNA. We find that two reduced suppressive between a p+ and different p- mutants can give very skewed mutants represent large deletions of the original hyper- results (5). Some p-s give rise to zygotic diploid clones, all suppressive p- genome with loss of the entire rep sequence of which contain p+ cells. Such a p- is said to be neutral with and all the other sequences required for ARS activity. The regard to zygotic suppressiveness. Other p-s produce some mutants, one neutral and one moderately suppressive, retain zygotic diploid clones that are entirely composed of p- cells. only 70 and 89 bp, respectively, from the p+ genome. These The fraction of zygotic diploid clones that are entirely p- is a contain only A T base pairs. characteristic of the particular p- mutant (6). A mutant that produces, for example, 20% p- diploid clones is said to be MATERIALS AND METHODS 20% suppressive. Strain KL14-4A/I21/HS3324 (a, hisi, trp2, leu2, p-), here Suppressiveness can be thought of as a consequence of called HS3324, and the growth media have been described competition between the amplified p- segment and the p' Abbreviations: kb, kilobase(s); bp, base pair(s); ARS, autonomous The publication costs of this article were defrayed in part by page charge replication sequence. payment. This article must therefore be hereby marked "advertisement" *Permanent address: Department of Genetics, SK-50, University of in accordance with 18 U.S.C. §1734 solely to indicate this fact. Washington, Seattle, WA 98195. 7156 Downloaded by guest on September 30, 2021 Genetics: Fangman and Dujon Proc. Natl. Acad. Sci. USA 81 (1984) 7157 Mbo Ava1i Nde I Ava 11 Mbo I bp (11) containing the rep2 sequence. Individual colonies of ijct Ois HS3324 were tested for reduced suppressiveness by crossing ,...... rp~ ~ " them on plates with a lawn of p+ cells. The zygotic diploids, selected by complementation of nutritional markers, form B I B patches that are replica-plated to glycerol plates to distin- 11110111i1111111111ilii S5 guish p+ and p- growth. Most patches, which contain many independent zygotes, give rise to only small papillae of growth, expected for the -98% suppressiveness of HS3324; FIG. 1. Map of HS3324 DNA. Map is arbitrarily linearized at the that is, -2% of the zygotes give rise to p+ clones, which can single Mbo I site. The map and the positions of the regions essential grow aerobically. An occasional patch produced confluent for ARS activity (I and B', or I and B) are taken from ref. 11. Arrow or nearly confluent growth on glycerol (Fig. 2a). In these labeledjct indicatesjunction between two excision points of HS3324 was and a subclone was mitochondrial DNA from the p+ genome. S4 and S5 are described in cases, the original colony purified, kesults. retested as large patches on a Petri dish (Fig. 2b). Approxi- mately 1% of the original HS3324 colonies were mutants by (11). A p0 derivative of HS3324 was obtained by growing these criteria. HS3324 in complete glucose medium with ethidium bromide Two of the spontaneous mutants, HS3324-S4 and HS3324- (20 ,g/ml). Suppressiveness tests used strain 777-3A/6 (a, S5 (here called S4 and S5), were analyzed further. Quantita- adel, pet9, p'). Media (2) and quantitative suppressiveness tive zygotic suppressiveness test results are shown in Table tests (12) have been described, except that zygotic colonies 1. The p+ tester culture contained 2.7% p- cells, as shown were scored for p' or p- phenotypes directly on unsupple- by the control mating to a p0 strain (a strain lacking mito- mented glucose plates after 10-14 days incubation at 28TC. chondrial DNA). These p- cells represent spontaneous p- p colonies remain small and white, while p+ colonies are mutants that arose during the cross or immediately before large and yellowish. Mitochondrial DNA was isolated by the mating, because the pet9 p+ tester culture cannot accumu- procedure described in ref. 13. late p- cells. Therefore, the results of each of the other mat- For yeast colony hybridization, portions of colonies were ings were adjusted for this background (31). Although S4 and picked from Petri dishes by laying a nitrocellulose sheet on S5 were isolated from the same culture, they appear to be the them for a few minutes. The inverted nitrocellulose sheet result of different . S4 is essentially neutral (1.5% was incubated consecutively, at room temperature, on paper suppressive), whereas S5 is still moderately suppressive at filter sheets saturated with the following: (i) 50 mM 35%. Tris HCl, pH 7.5/25 mM sodium EDTA/1% 2-mercapto- A clone with reduced suppressiveness could actually be a ethanol/500 mM NaCl (30 min); (ii) solution i containing Zy- heterogeneous population of cells in which two kinds of molyase 60,000 (Seikagaku Kogyo, Tokyo) (65 min); (iii) 500 mitochondrial genomes are segregating (1), one the original mM NaOH (8 min); (iv) 1 M Tris HCl, pH 7.8 (2 min); (v) 500 hypersuppressive and the other a neutral variant or even a mM Tris HCl, pH 7.8/1.5 M NaCl (2 min, and then repeated p°. This possibility was tested with S5, which would have to for 2 min). (Subsequent experiments have shown that step ii, be segregating an appreciable fraction of both neutral and the treatment with Zymolase, is not necessary.) The filter hypersuppressive cells in the culture. Forty-five colonies was air dried, then baked at 80'C for 2 hr. Before hybridiza- were streaked and tested by mating to a lawn of p+ tester tion, the filter was prehybridized for 30 min at 650C (repeat- cells. All 45 gave confluent growth on glycerol and all had, ed once with fresh solution) with 10 ml of hybridization solu- therefore, reduced suppressiveness. This test demonstrates tion per 8-cm-diameter filter. Hybridization was carried out the absence of the original hypersuppressive p-, but it does overnight at 650C in 1.2 M NaCl/0.12 M Na citrate/0.1% not distinguish between a homogeneous clone of a reduced bovine serum albumin/0. 1% polyvinylpyrrolidone/0. 1% Fi- suppressive mutant and a clone that also accumulates p0 coll/0.1% NaDodSO4/100 ,ug of denatured herring sperm cells. This last possibility was eliminated by colony - DNA per ml, and 32P-labeled nick-translated probe. Mitochondrial DNAs from mutants S4 and S5 were a FIG. 2. Plate screening tests sheared by sonicating 50 gg of DNA in 200 ,ul of 10 mM for reduced suppressive mu- Tris *HCl, pH 8/0.1 mM EDTA in a 1.5-ml Eppendorf Micro- tants. (a) Sixty-four colonies of fuge tube. The sonication time and power was such that an HS3324 were transferred to an 8 equivalent amount of X DNA was sheared to a mean size of x 8 grid on a Petri dish and were -1 kb. Treatment with Klenow polymerase I, addition of crossed by replica plating to a BamHI linkers, ligation into the BamHI site of pBR322, and lawn of tester cells on an unsup- transformation of E. coli HB101 followed standard proce- plemented glucose plate. After 2 dures (14). Recombinant carrying S4 or S5 mito- days at 28°C, the patches of zy- gotic diploid growth were repli- chondrial DNA were detected by hybridization. For se- ca-plated to glycerol-rich medi- quencing, the plasmids containing the cloned DNAs were um (N3) plates and examined af- cleaved with Msp I. The largest fragments, containing the ter a further 2 days of growth. BamHI linked inserts, were purified on gels, end-labeled (b) Colonies that appeared to with [y-32P]ATP and polynucleotide kinase. The labeled have reduced suppressiveness fragments were cleaved within the pBR322 sequences with a b were streaked out and sub- second . For S4, after Taq I digestion, the clones were retested as large =z900-bp Taq I/Msp I piece was isolated and partially se- patches. Lower left, HS3324 quenced according to ref. 15. The first 12 bases are pBR322 control cells. In the remaining the I quadrants are three subclones sequences. For S5, Msp fragment was cleaved with from patches that appeared to EcoRV and the mixture of the two fragments (15 bp and be reduced suppressives in the -1050 bp) was sequenced directly. The first 15 bp are a mix- first test. Two continue to ap- ture of pBR322 sequences. pear as reduced suppressives and one does not. The two mu- RESULTS tants were further analyzed by Isolation of Reduced Suppressive Mutants. Strain HS3324 quantitative suppressiveness contains a hypersuppressive p- mitochondrial DNA of 963 tests. Downloaded by guest on September 30, 2021 7158 Genetics: Fangman and Dujon Proc. NatL Acad ScL USA 81 (1984) Table 1. Quantitative zygotic suppressiveness tests GC GC F++ Diploid colonies GATC GATC scored % Haploid Respiratory respiratory % 3 parent Total deficient deficient suppressive 3 HS3324 455 447 98.2 98.2 2 S4 1501 63 4.2 1.5 2! S5 808 299 37.0 35.2 p0 1405 38 2.7

ization using cloned HS3324 DNA as probe. No p0 colonies *-ww- were found among 60 colonies of HS3324, S4, or S5 that were tested (Fig. 3). The 60 colonies arose from the same population of cells used in the quantitative tests of Table 1. I We conclude, therefore, that S4 and S5 are homogeneous

clones of reduced suppressiveness. The colony hybridiza- . _ tion results demonstrate three additional points. First, the hybridization signal is uniform among subclones of a strain, S5 FIG. 4. Sequencing gels of suggesting that there is little variation in mitochondrial DNA S4 and S5 mitochondrial DNAs. content among them. S4 and S5 colonies a sub- Arrows delimit successive repe- Second, gave titions (1-3), 70 bp each for S4 stantially reduced hybridization signal compared to HS3324 and 89 bp each for S5. Other gel colonies. Third, S4 and S5 gave significant differences in hy- loadings, not shown, allowed bridization signals; S4, which is neutral, showed less hybrid- the sequences to be clearly read ization than S5, which is a moderately suppressive mutant. through three successive re- Analysis of Mitochondrial DNAs. The reduced colony hy- S4 peats in each case. bridization signal for S4 and S5 (Fig. 3) must mean that either these mutants contain fewer copies ofthe HS3324 genome or necessary to clone randomly sheared DNA fragments for se- they represent large deletions of the HS3324 DNA. Prepara- quencing. DNA preparations were sonicated to decrease the tions of total nucleic acid from the strains were centrifuged size of most of the DNA to <2 kb. Molecules of 700-900 bp to density equilibriumn in CsCl/Hoechst 33258 solutions. All were isolated from preparative gels and treated with Klenow three strains produced bands of mitochondrial DNA of com- polymerase to fill in any protruding 5' ends. BamHI linkers parable intensity relative to nuclear DNA. Therefore, S4 and were added and the molecules were ligated into the BamHI S5 appear to be deletion variants of HS3324 that have ampli- site of pBR322. Inserts of S4 and S5 DNA were detected by fied the remaining DNA. hybridization of nick-translated S4 or S5 DNAs to trans- Purified S4 and S5 mitochondrial DNAs were tested for formed E. coli colonies on ampicillin plates. Sequencing was cleavage with all restriction enzymes that have sites (17 to- carried out as described, using plasmids pS4M-1 and pS5M- tal) in the 963-bp HS3324 DNA (11). No cleavage occurred 4, which contain -850-bp inserts of S4 DNA and S5 DNA, (data not shown), indicating that all sites are deleted or al- respectively. tered. The S4 and S5 sequences were localized approximate- Sequencing (15) shows that both S4 and S5 consist entirely ly on the HS3324 DNA by using them as 32P-labeled nick- of A T base pairs (Fig. 4). The cloned DNAs consist of tan- translated probes against restriction endonuclease-cleaved dem direct repeats of a 70-bp sequence for S4 and an 89-bp fragments of HS3324 DNA. Both S4 and S5 could be local- sequence for S5. As expected from the hybridization results, ized with no ambiguity to a 173-bp Ava II/Nde I fragment both sequences are derived from the 173-bp Ava II/Nde I (data not shown; see Fig. 1), a fragment with no known re- fragment of HS3324 (Fig. 5). S4 and S5 share 67 bp of se- striction sites. quence, but both their left and right end points are different. Gel electrophoresis of purified S4 and S5 mitochondrial Therefore, they arose by distinct excision events. S5 excised DNAs revealed a broad distribution of ethidium bromide- within two 5-base sequences of A-T-A-T-A and S4 excised staining material ranging in mobility from zero (material re- within two 7-base sequences of A-T-A-T-A-T-A (Fig. 5). The tained in the wells) to that expected for linear DNA of <200 excision event for S4 was formally equivalent to an unequal bp; a closely spaced band pattern, which may represent a cross-over occurring within two (A-T)7 sequences creating multimeric circle series as in other p- mutants (see ref. 16 for an (A-T)j, run; at least four of the A-T sequences came from a review), was barely visible (data not shown). The absence each end point. The full tract of homology between the two of restriction endonuclease sites in S4 and S5 DNAs made it excision points in HS3324 extends 25 bp for S4, and 19 bp HS3324 S4 S5

**.*.s:...... *.*...... * 40l * #41 4 "m .0 * ". 0 o * _ 4 * A

*e ' *.t. lot, O* .. tf

*r$ ***I*", V * - .#*..W~ *1-.4 9, U_ 4A* 4' 4 b *

FIG. 3. Yeast colony hybridization. Colonies representing subclones of strains HS3324, S4, and S5 were probed with nick-translated pSCM128 (8), pBR322 containing the complete HS3324 DNA. Each set of 64 colonies includes 4 colonies, forming the lower left diagonal, as a negative control. The three filters were hybridized in the same solution and exposed for the same length of time. Downloaded by guest on September 30, 2021 Genetics: Fangman and Dujon Proc. Natl. Acad. Sci. USA 81 (1984) 7159

25 25

19 '650 '.700 mS4m750 AATATATAAA TMTATAAAT ATATTATATA TATAATATAA TATATATATA TATAAAATAA TAAATTATAT A'ATATAATAT ATATATATAT MTAATAAAMT TATATATATA TATATAAAAT AATAAAAATA 16. 5016 2716'.:16 1 27 -:27

FIG. 5. Sequences of S4 and S5 in the HS3324 genome. Only 130 of the 173 bp of the Ava II/Nde I fragment of HS3324 (11) are shown, with the sequences retained in S4 and S5 overlined and underlined, respectively. Open circles indicate uncertainty of excision points for S4 and S5 within the 7-bp and 5-bp repeated elements, respectively. The 650th, 700th, and 750th bases from the unique Mbo I cleavage site in HS3324 DNA are indicated. The closest G-C base pairs are at positions 613 and 756. The closest boundary of the 300-bp rep2 sequence is at position 612. Above the sequence, horizontal lines with arrows indicate the 25-bp direct repeats within which the S4 excision event occurred, and the 19- bp direct repeats (containing two mismatches marked x) within which the S5 excision took place. Below the sequence, horizontal lines with arrows indicate three other sets of direct repeats, two of 16 bp and one of 27 bp. with one mismatch on each side of the A-T-A-T-A sequence Requirements for Replication. Segments from many parts for S5 (Fig. 5). Excisions from the p+ genome often occur in of the p+ genome, when cloned into plasmids, promote ARS such short 100% A+T regions (17). The position of the S4 activity in yeast (20). However, because hybrid plasmids ap- and S5 sequences within the 963 bp of HS3324 DNA and the pear to reside in the nucleus (8, 21), the relevance of this regions essential for ARS activity in an E. coli-yeast plasmid property to mitochondrial replication is uncertain. Deletion are shown in Fig. 1. analysis of cloned HS3324 DNA has recently shown (11) that ARS activity requires a small, palindromic G+C-rich region, DISCUSSION called region I (Fig. 1), which is part of the rep2 sequence The structure of the S4 and S5 genomes is relevant to ques- and one of two A+T-rich regions (here called B and B'; Fig. tions about the relationship of genome size and suppressive- 1) in the flanking unique DNA (11). Experiments with plas- ness, about the relationship of ARS activity to replication in mids reconstructed from various deletion derivatives have the mitochondrion, about minimal sequence requirements led to the suggestion that ARS activity actually requires, at for both replication and suppressiveness within the mito- an appropriate distance from the I region, one of six 11-base chondrion, and about the formation and maintenance of ex- consensus sequences found in HS3324 DNA (11). The con- tremely short mitochondrial genomes. Requirements for Suppressiveness. From earlier work (7, sensus sequence, 5' _-A-A-A-c-A-T-A-A-A-A 3', is one that 9), it is known that hypersuppressiveness is limited to those is common to several ARS elements (22, 23). S4 and S5 p strains that contain one of the rep sequences and have a DNAs do not retain the I region or any of the six perfect 11- short repeated sequence. p-s containing a rep within long base consensus sequences (Figs. 1 and 5), although se- sequences are not hypersuppressive. And p-s that lack a rep quences closely related to the consensus are abundant. Al- sequence are not hypersuppressive, even if they consist of a though the S4 and S5 DNAs appear to replicate faithfully in short repeat. However, the structure of S5 DNA shows that the mitochondria because the genomes are stably maintained considerable suppressiveness (35%) can be accomplished (Fig. 3) at the normal cellular concentration, a deletion deriv- without a rep sequence and without G C base pairs. Assum- ative (pAGT363) of cloned HS3324 DNA that contains the ing that the 89-bp sequence retained by S5 occurs only once entire S4-S5 sequences does not have ARS activity (11). in the 75-kb p+ genome, it is present in an S5 cell at -850 From these observations, one might conclude that ARS ac- times its concentration in a p+ cell. The difference in sup- tivity does not necessarily reflect the ability to replicate pressiveness of S4 and S5 may be accounted for by differ- within the mitochondrion. However, ARS activity was as- ences in the sequences retained from HS3324, by repeat size sessed with plasmids that contain only a single copy of the per se, or by the sequences created by new junctions in the S4-S5 region, whereas the S4 and S5 sequences are present DNAs. Another neutral p- mutant, RD1A, consists of a 68- in the mitochondrion as large head-to-tail multimers. Recent bp DNA repeat and contains only two G-C base pairs (18, experiments show that the cloned "800-bp segments of S4 19). A sequence comparison of S4, S5, and RD1A shows and S5 DNA, which consist of -10 tandem repeats of the 70- several regions of sequence identity with a maximum stretch bp or 89-bp sequences, have ARS activity. of 16 bp (T-A-T-A-T-A-A-T-A-T-A-A-T-A-T-A). On a ran- The S. cerevisiae mitochondrial genome is 82% A+T and dom basis, this 16-bp sequence is expected only once in the contains long stretches that are, or are nearly, 100% A+T. 75,000-bp p+ mitochondrial genome. The S4-S5 region of Known genes as well as other open reading frames can be HS3324 shows a 27-bp direct repeat (Fig. 5), only one copy located on the p+ genome, in part by their marked enrich- being present in S4 and two copies in S5. The 16-bp homolo- ment in and regular distribution of G *C base pairs, whereas a gy with RD1A occurs precisely within this 27-bp repeat (be- number (50-100) of short G+C-rich clusters of unknown ginning at the 5' boundaries shown in Fig. 5). This common function lie conspicuously in A+T-rich intergenic regions or sequence is present every 68 bp in RD1A, every 70 bp in S4, even at specific points inside reading frames (24, 25). A few and is separated by 37 bp and 52 bp in S5. The difference in of the clusters (rep or ori sequence) have been proposed to the frequency of this sequence may explain the difference in be origins of replication, although a direct functional test has suppressiveness between S5 and S4 or RD1A. not yet been devised. Others of these G+C-rich clusters Four additional, independently occurring, reduced sup- have been proposed to be sequences that act as surrogate pressive mutants have been isolated from HS3324. All of origins in the absence of all the normal origins (e.g., see ref. them map to the 173-bp Ava II/Nde I fragment. Two are 26). However, the existence of the S4 and S5 genomes defin- neutral and two are moderately suppressive. Sequence anal- itively shows that not a single G-C base pair is necessary for ysis of these mutants should help determine whether it is size DNA to replicate in the mitochondrion. Efficiencies of repli- or sequence that is important for suppressiveness. cation, however, have not yet been compared. An individual Downloaded by guest on September 30, 2021 7160 Genetics: Fangman and Dujon Proc. NatL Acad Sci. USA 81 (1984) S4 (or S5) sequence may permit only very inefficient initia- ing the establishment of new p- genomes, and (iii) exhibit tion of replication compared to other (G+C-rich) sequences, moderate suppressiveness. the inefficiency being compensated for by the existence of W.L.F. thanks B.D., Alain Jacquier, Hugues Blanc, and Franqois many repeats in a single molecule. Michel for sharing ideas, expertise, and reagents, and for making his If S4 and S5 contain a specific sequence essential for repli- stay in their laboratory enjoyable; Dr. B. Brewer for many helpful cation, that sequence might be found in other p- molecules discussions of the work; Drs. B. Brewer, C. Newlon, and R. Scla- as well. The 68-bp RD1A mitochondrial DNA, as discussed fani for criticism of the manuscript; and Nancy Gamble for prepara- above, contains a 16-bp identity with S4 and S5 (T-A-T-A-T- tion of the paper. This work was supported by the "ATP" program A-A-T-A-T-A-A-T-A-T-A), making it a candidate for a repli- of the Centre National de la Recherche Scientifique, by a grant from cation origin. Further comparisons with the published com- Institut National de la Sante et de la Recherche Mddicale (PRC plete sequences of other p- DNAs may allow us to identify a 134004) to B.D., and by a U.S. Public Health Service grant (GM18926) common essential element, if one exists. One might suppose, to W.L.F. on the contrary, that the small highly A+T-rich genomes of 1. Dujon, B. (1981) in The Molecular Biology of the Yeast Sac- S4, S5, and RDIA are able to replicate in the mitochondrion charomyces cerevisiae, eds. Broach, J., Jones, E. & Strathern, simply because their high A+T content, perhaps by facilitat- J. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), ing strand unwinding, allows an increased affinity for the pp. 505-635. that 2. Coen, D., Deutsch, J., Netter, P., Petrochilo, E: & Slonimski, replication initiation machinery. In fact, it is possible P. (1970) Symp. Soc. Exp. Biol. 24, 449-496. replication initiation normally takes place at A+T-rich, but 3. Lukins, H. B., Tate, J. R., Sanders, G. W. & Linnane, A. W. sequence nonspecific, regions of the p+ genome. (1973) Mol. Gen. Genet. 120, 17-25. At present, there is no way to directly compare the effi- 4. Waxman, M. F. & Birky, C.W., Jr. (1982) Curr. Genet. 5, 171- ciencies of replication in the mitochondrion of unit genomes 180. as different as the 75-kb p+ DNA, the 963-bp HS3324 DNA, 5. Ephrussi, B., de Margerie-Hottinguer, H. & Roman, H. (1955) and the 70-bp S4 DNA. The in vivo tests used so far assume Proc. Natl. Acad. Sci. USA 41, 1065-1071. that the degree of suppressiveness reflects replication effi- 6. Ephrussi, B., Jakob, H. & Grandchamp, S. (1966) Genetics 54, ciency. However, segregation must still be considered as an 1-29. 7. de Zamaroczy, M., Marotta, R., Faugeron-Fonty, G., Gour- explanation, in whole or in part, for suppressiveness. p se- sot, R., Mangin, M., Baldacci, G. & Bernardi, G. (1981) Na- quences that are more suppressive could be those that are ture (London) 292, 75-78. more efficiently segregated. 8. Blanc, H. & Dujon, B. (1982) in Mitochondrial Genes, eds. Excision of Extremely Short Mitochondrial Sequences. Be- Slonimski, P., Borst, P. & Attardi, G. (Cold Spring Harbor cause excision points of many p- mutants occur in short di- Laboratory, Cold Spring Harbor, NY), pp. 279-294. rect or inverted repeats, it has been suggested that p- dele- 9. Blanc, H. & Dujon, B. (1980) Proc. Natl. Acad. Sci. USA 77, tions arise by intramolecular recombination between short 3942-3946. repeats in the p+ genome (27-29). Mutants such as S4 and S5 10. Dujon, B. & Blanc, H. (1980) in The Organization and Expres- appear to have excised from very closely spaced sites. While sion of the Mitochondrial Genome, eds. Kroon, A. M. & Sac- cone, C. (Elsevier/North-Holland, Amsterdam), pp. 33-36. the required DNA cyclization event for such short DNA seg- 11. Blanc, H. (1984) Gene, in press. ments has ah extremely low probability in solution (30), cy- 12. Ephrussi, B. & Grandchamp, S. (1965) Heredity 20, 1-7. clization in vivo may be facilitated by . Alternative- 13. Jacquier, A. & Dujon, B. (1983) Mol. Gen. Genet. 19, 487-499. ly, unequal intermolecular recombination could create tan- 14. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular dem repeats of the short sequences and thereby increase the Cloning: A Laboratory Manual (Cold Spring Harbor Labora- probability of cyclization and excision. The high density of tory, Cold Spring Harbor, NY). long (¢16 bp) direct repeats in the S4-S5 region (Fig. 5) may 15. Maxam, A. M. & Gilbert, W. (1980) Methods Enzymol. 65, greatly increase intra- and intermolecular recombination 499-560. events, accounting for the fact that all six reduced suppres- 16. Locker, J., Lewin, A. & Rabinowitz, A. (1979) Plasmid 2, 155- 181. sive p- mutants are derived from this region. The density of 17. de Zamaroczy, M., Faugeron-Fonty, G. & Bernardi, G. (1983) direct repeats is lower in other parts of HS3324 sequence. Gene 21, 193-202. Excision might also occur via abortive replication in the 18. Moustacchi, E. (1972) Biochim. Biophys. Acta 277, 59-60. A+T-rich region, which includes S4 and S5. For example, 19. Van Kreijl, C. F. & Bos, J. C. (1977) Nucleic Acids Res. 4, shortly after replication is initiated, the two new strands 2369-2388. could sometimes be expelled from the parental molecule by 20. Hyman, B., Cramer, J. H. & Rownd, R. H. (1984) Gene 26, branch migration. A short duplex produced in this way is 223-230. likely to have 3' single-strand tails as a result of a leading/ 21. Hyman, B., Cramer, J. H. & Rownd, R. H. (1982) Proc. NatI. lagging strand replication mechanism. The two tails would Acad. Sci. USA 79, 1578-1582. short 22. Stinchcomb, D. T., Mann, C., Selker, E. & Davis, R. W. facilitate circularization, because they usually contain (1981) in The Initiation ofDNA Replication, ICN-UCLA Sym- complementary runs of alternating adenines and thymines. posium on Molecular and Cellular Biology, eds. Ray, D. S. & Fox, C. F. (Academic, New York), Vol. 22, pp. 473-488. 23. Broach, J. R., Li, Y.-Y., Feldman, J., Jayaram, M., Abraham, CONCLUSION J., Nasmyth, K. A. & Hicks, J. B. (1983) Cold Spring Harbor Symp. Quant. Biol. 47, 1165-1174. From the results presented here, and in keeping with most 24. Hudspeth, M. E. S., Vincent, R. D. Perlman, P. S., Shu- earlier observations on p- mutants, we think it is likely that mard, D. S., Treisman, L. 0. & Grossman, L. I. (1984) Proc. the mitochondrial DNAs in mutants S4 and S5 consist of a NatI. Acad. Sci. USA 81, 3148-3152. single sequence (70 and 89 bp, respectively) repeated many 25. Michel, F. (1984) Curr. Genet. 8, 307-318. times. A cell would contain -20,000 copies of the repeat 26. Goursot, R., Mangin, M. & Bernardi, G. (1982) EMBO J. 1, we three 705-711. unit. However, since have sequenced only repeats 27. Gaillard, C., Strauss, F. & Bernardi, G. (1980) Nature (Lon- for each mutant we cannot eliminate the possibility that se- don) 283, 218-220. quence heterogeneity exists. Assuming sequence homogene- 28. Bernardi, 0. (1982) Trends Biochem. Sci. 7, 404-408. ity, the results presented here allow a simple conclusion 29. Sor, F. & Fukuhara, H. (1983) Cell 32, 391-396. about yeast mitochondrial DNA: No G-C base pairs are re- 30. Shore, D. & Baldwin, R. L. (1983) J. Mol. Biol. 170, 957-981. quired for a DNA molecule to (i) faithfully replicate and seg- 31. Nagley, P., Gingold, E. B., Lukins, H. B. & Linnane, A. W. regate within the mitochondrion, (ii) excise and amplify dur- (1973) J. 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