Proc. Natl. Acad. Sci. USA
Vol. 73, No. 9, pp. 3073-077, September 1976 Biochemistry
Fate of mismatched base-pair regions in polyoma heteroduplex DNA during infection of mouse cells (genotypic markers/restriction endonucleases/repair/recombination/replication)
Lois K. MILLER*, BEVERLEY E. COOKE, AND MIKE FRIED Imperial Cancer Research Fund, P.O. Box 123, Lincoln's Inn Fields, London WC2A 3PX, England
Communicated by Renato Dulbecco, June 16,1976
ABSTRACT Heteroduplex DNA has been constructed from two variants of polyoma virus that differ genotypically at four distinct sites. The genotypes of the progeny virus derived from infections of mouse cells with single heteroduplexes have been analyzed to determine how the genotypic markers of the parental heteroduplex segregate. Markers that are separated by a length of DNA greater than 600 nucleotides segregate independently. Segregation was not detected between two markers separated by only approximatel 90 nucleotides. We interpret these results on the basis of the correction of mismatched base-pair regions in the heteroduplex before the completion of DNA replication. We suggest that this technique provides valuable information concerning gene conversion in mammalian cells and permits the transfer of genotypic markers from one virus strain to another.
During experiments involving construction of a genetic map of the small, double-stranded DNA genome of polyoma virus (1, 2), we became interested in determining whether heteroduplex polyoma DNAs containing mismatched base-pairs are first replicated, resulting in the independent segregation of two DNAs of distinct genotypes, or whether other cellular mechanisms correct the mismatched base-pair region before the completion of DNA replication, resulting in DNA of one genotype only. The question was generally interesting with regard to DNA transitions in mammalian cells. The question was also important with respect to viral genetics in predicting the genotype of progeny virus produced from heteroduplex DNA infections in genetic mapping experiments involving the marker rescue technique (Hewick, Waterfield, Miller, and Fried, manuscript in preparation). Correction of mismatched base-pairs in heteroduplex DNA has been studied extensively in bacteria using heteroduplex phage DNA (3-8). These studies demonstrated that cellular mechanisms are present in prokaryotes that can correct (repair) mismatched base-pair regions in heteroduplex DNA before the completion of DNA replication. In OX174, the correction of a mismatched base-pair before replication depended on the position of the mismatched base-pair relative to the origin of DNA replication (4); the closer the mismatched base was to the origin, the less frequently it was corrected before DNA replication. Correction of mismatched base-pairs may involve four basic steps, similar to those described for the repair of UV-damaged DNA (9, 10). First, the mismatched base-pair may be recognized and marked by an endonucleolytic nick (single-strand break) in one of the two DNA strands. Second, the nucleotide Abbreviations: FI DNA, supercoiled circular DNA; FII DNA, relaxed (nicked) circular DNA, i.e., DNA containing a single-strand break; FEI DNA, linear DNA; EcoRI, restriction endonuclease RI from Escherichia coil; HhaI, HaeIII, HindIII, and HpaII, restriction endonucleases from Haemophilus haemolyticus, H. aegyptius, H. influenzae, and H. parainfluenzae, respectively; TS, temperature sensitive. *
Present address: Department of Bacteriology and Biochemistry, The University of Idaho, Moscow, Idaho 83843. 3073
sequences around this nick on the DNA strand may be degraded by exonuclease action, forming a single-stranded gap. This can be followed by DNA synthesis, which fills the gap by the action of DNA polymerase using the opposite strand sequences as a template. Finally, the end of a newly synthesized sequence can be joined to preexisting sequences on the repaired strand by the action of DNA ligase. Although it is known that mammalian cells possess an excision-repair system for correction of UV-damaged DNA (11, 12), information concerning the ability of mammalian cells to correct mismatched base-pairs is lacking. Studies of gene conversion and postmeiotic segregation in fungi and Drosophila suggest the formation of segments of heteroduplex DNA. As
an intermediate step in the recombination process with mismatched base-pair regions within the heteroduplex DNA segment corrected (13-17). -Mammalian systems may also contain enzymes for correction of mismatched base-pair regions. The results of Lai and Nathans (18) using heteroduplex DNA of simian virus have been explained on the basis of correction of mismatched base pairs in African green monkey cells. In order to answer the question of whether mismatched base-pairs of heteroduplex DNA were corrected before DNA replication occurred, we used a unique set of silent mutations of polyoma virus. These silent mutations are detectable by restriction endonuclease analysis, but they have no effect on the phenotype of the viruses. Each strand of the viral heteroduplex DNAs constructed contained four distinguishable genotypic markers, and the segregation of these markers in the progeny viruses could be monitored. MATERIALS AND METHODS Viruses and Preparation of Viral DNAs. The TS (temperature sensitive)-A variant (19) and the CR variant (Fried, unpublished results) were isolated from the Pasadena large plaque strain of polyoma virus. Virus stocks were derived from single plaque isolates and passaged at low multiplicity. Supercoiled viral DNA was purified from Hirt (20) supernatants of infected mouse embryo cells by CsCl/ethidium bromide equilibrium density centrifugation followed by sedimentation in neutral sucrose gradients C21). Heteroduplex Formation. The TS-A F1 DNA (supercoiled circular DNA) was randomly nicked by x-irradiation so that 30% FI was converted to FII (nicked circular DNA). The singly-nicked FII DNA was purified from the remaining FI by CsCl/ethidium bromide equilibrium density centrifugation (22). The CR FI DNA was cleaved into two fragments by incubation with HindIll restriction nuclease (from Haemophilus influenzae) at 370 in 10 mM Tris-HCl (pH 7.4)-6 mM MgCI2-1 mM dithiothreitol-50 mM NaCl. The enzyme was heat-denatured by incubation at 80° for 15 min. The TS-A FII DNA (1.1 ,ug/ml) and HindIII-cleaved CR DNA (11 ,g/ml) were denatured in 0.4 M NaOH at room temperature for 20 min. The
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Proc. Nati. Acad. Sci. USA 73 (1976) CR
Hindill
Hhai 0 "-
Infect cells at 390C The "a" Phenotype is selected
Analyze Genotype of Progeny from Single Heteroduplex
FIG. 1. The experimental design. Two variants of polyoma, TS-A and CR, differ genotypically at four distinct sites (A versus a, B versus b, C versus c, and D versus d). The approximate position of genotypically different sites (0, *, X, *) are marked relative to the origin of bidirectional, semiconservative DNA replication (OR). Heteroduplexes between TS-A and CR DNAs are formed by denaturation and reannealing of nicked parental DNAs (see Materials and Methods). Secondary whole mouse embryo cells are infected with heteroduplex DNAs. Plaques are developed at 38.50 to select for the "a" phenotype. The DNAs derived from individual plaques are analyzed for the genotype markers Bb, Cc, and Dd by digestion with restriction nucleases (see Materials and Methods).
solution was neutralized with HCl and buffered by 0.16 M sodium phosphate buffer (pH 7.0) and the strands were reannealed at 68° for 30 min. The DNA solution was diluted with Dulbecco's phosphate-buffered saline solution minus Mg++ and Ca++. Infection of Cells with Heteroduplex DNA. DNA was spread on mouse embryo cells and the cells were incubated (1). After 8 days at 38.50 the cells were stained with nbutral red and plaques picked the following day. Virus stocks were prepared at 38.50 from each individual plaque. Approximately 1 X 104 plaque-forming units/.ug of heteroduplex DNA appeared at 38.50. Less than 1 X 102 plaque-forming units/Ag appeared on plates containing TS-A without CR HindIll-digested DNA. The CR HindIII fragments alone produced plaques at one-ninth the frequency of CR HindIII fragments with TS-A FII in heteroduplex form described above. Genotypic Analysis with Restriction Nucleases. Supercoiled FI DNA was prepared as described above from infection of whole mouse embryo cells with virus stocks derived from single plaques. The polyoma FI DNAs were digested with endonuclease HpaII (from H. parainfluenzae), HhaI (from H. haemolyticus) or HaeIII (from H. aegyptius) in 10 mM TrisHCl (pH 7.5)-10 mM MgCl2-1 mM dithiothreitol. For each enzyme digestion, approximately 2 Mg of FI DNA were digested at 370 for approximately 3 hr with sufficient enzyme for complete digestion. The DNA fragments were then analyzed on 20 X 20 cm slab gels. For DNA digested with HhaI and HaeIII, 1.4% agarose slab gels were used. For HpaII-digested DNA, 3.3% acrylamide-0.17% bisacrylamide-0.5% agarose gels
FIG. 2. The physical map of polyoma DNA indicating the location of the EcoRI, HinHI, HhaI, and HpaII restriction endonuclease cleavage sites, the origin (0) and termination of DNA synthesis (adapted from ref. 14, and Griffin, personal communication). The circular polyoma DNA genome is composed of approximately 5200 nucleotide pairs. The map is divided into 100 map units with the EcoRI cleavage site at 00 map units. The TS-A is cleaved by HhaI at the three sites indicated; the CR variant lacks the HhaI cleavage site at 26.4 map units (denoted as B or b in Fig. 1). HaeIll cleaves CR approximately 24 times. Only one of these cleavage sites is denoted on the map (HaeIII*) at 28.4 map units. The TS-A variant lacks this HaeIII site (denoted as C or c in Fig. 1).
were used. The restriction nuclease fragment patterns were visualized by ethidium bromide staining.
RESULTS Differences Between the TS-A and CR Variants. The experimental design for studying the repair of polyoma heteroduplex DNA is schematically presented in Fig. 1. Heteroduplexes were formed between the two polyoma variants, TS-A and CR, which differ genotypically in four distinct sites on the genome. One site is a mutation that governs the ability of the variant strains to produce infectious virus at 38.50. The CR strain will form plaques at 38.50, whereas the TS-A strain will not. The position of the mutation responsible for this temperature-sensitive (TS) property of TS-A has been located by a marker rescue technique (1) and is located between the Hindfll restriction enzyme cleavage site at 1.0 map unit and the Hha I restriction enzyme cleavage site at 14.0 map units on the polyoma physical map (see Fig. 2). This site has been designated as A for TS-A and "a" for CR (see Fig. 1). A second site where the two variants differ is at the HhaI site at 26.4 map units on the polyoma physical map (Fig. 2). The TS-A variant contains the Hha cleavage site, whereas the CR variant does not (C. Barry and M. Griffiths, personal communication). Thus, when TS-A DNA is digested with HhaI, three fragments (46, 42, and 12%) are generated (27), whereas HhaI digestion of CR DNA results in the generation of only two fragments (58 and 42% of the genome) (see Fig. 3). This site at 26.4 map units (Griffin, personal communication) has been designated B for TS-A and b for CR (Fig. 1). The two variants also differ at another restriction enzyme cleavage site. This is at the HaeIII site at 28.2 map units on the physical map (Fig. 2; Griffin, personal communication) joining HaeIII fragment 6 and HaeIII fragment 20. The TS-A DNA, lacks this restriction enzyme site, resulting in the absence of these two fragments and the appearance of a new fragment (see Fig. 4). The new fragment migrates in gels to a position expected for a molecule of molecular weight equal to the com-
Biochemistry:
Miller et al. A
B
Proc. Natl. Acad. Sci. USA 73 (1976)
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TS-A A2
C
u_ ...
do
406
FIG. 3. HhaI restriction endonuclease fragment patterns of TS-A and CR DNAs. FI DNA of TS-A and CR DNA were digested with HhaI restriction endonuclease and the fragments were electrophoresed through 1.4% agarose gels in the presence of ethidium bromide. Photographs of the gels were taken under ultraviolet illumination; the negative print is shown above. (A) Control FIT, FIII (linear), and FT of undigested A2 DNA. (B) HhaI-digested TS-A DNA. (C) HhaT-digested CR DNA; arrows on right point to the TS-A fragments missing in the CR fragment pattern. Note the new fragment in the CR pattern, which is a combination of the two TS-A fragments.
bined molecular weight of HaeIll-6 and HaeTIT-20 fragments. This HaeIII site is designated C in TS-A and c in CR (Fig. 1).
The fourth site where the two variants differ is located in HpaTT-5, near the origin of DNA synthesis. HpaII-5 differs in size between different isolates of the Pasadena large plaque strain by approximately 0.3% of the genome or 18 base pairs (23). This difference is located between 70.7 and 72.7 map units of the polyoma physical map (Griffin, personal communication) (Fig. 2) and can easily be detected by the electrophoretic mobility of HpaII-5 in polyacrylamide gels (23}. The TS-A strain contains the more slowly migrating, or larger, Hpall-5. This site is designated D for TS-A and d for CR (Fig. 1). Formation of Heteroduplex DNA. Since the DNA strands of polyoma are difficult to separate, the method of heteroduplex formation was carefully designed and selection was applied to allow us to study only the heteroduplexes rather than the homoduplexes. To reduce the amount of infectious CR homoduplexes, we cleaved the CR FT DNA with HindIII, which cleaves the DNA twice: one cut is at 1.0 map unit, the other is at 45.0 map units (24). This not only permitted separation of strands under alkaline conditions, but also reduced the formation of infectious CR homoduplex DNA. To select against the TS-A homoduplexes, we isolated the virus analyzed from plaques formed at 38.5°, a temperature at which the TS-A homoduplexes will not form plaques. Thus, all the virus isolates studied, being selected at 38.5°, have the "a" genotype (see Tables 1 and 2). To favor heteroduplex formation, a 10-fold molar excess of CR HindITI fragments were added to the TS-A FIT DNA. After denaturation and renaturation (see Materials and Methods) the majority of the circular DNA molecules are heteroduplexes composed of one CR DNA strand with two nicks at HindITT sites and one TS-A DNA strand with one random nick in half of the heteroduplexes.. Under the conditions used the number of plaques appearing due to the
FIG. 4. The HaellI restriction nuclease fragment patterns of TS-A and A2 DNAs. 32P-Labeled FI DNA was prepared from 3T6 cells infected with TS-A or A2 viruses (24). Fl DNAs were digested with HaeIII (see Materials and Methods) and the fragments were electrophoresed through 7% polyacrylamide gels (20 X 40 cm). Autoradiographs of the resulting fragment patterns of the two DNAs are shown above. The arrow on the left points to the new fragment of TS-A, which is a combination of two smaller fragments seen in the A2 pattern marked by arrows on the right. With respect to these fragments, CR DNA has a similar HaeIII cutting pattern as A2 DNA.
reannealing of CR HindHI fragments is one-ninth the amount of plaques developed for CR/TS-A heteroduplexes. Since we know the genotype of the CR HindIII homoduplexes is abed, we can subtract out one abcd genotype for every nine plaques studied and thus correct for the contaminating level of CR homoduplex (Table 1). A diagrammatic representation of the heteroduplex formation with the location of the genotypic markers is shown in Fig. 1. Genotypic Analysis. Viral DNAs derived from 18 plaque isolates of heteroduplex DNA were analyzed by restriction enzymes (see Materials and Methods) and classified according to genotype (Table 1). There are four important observations to note from these results: (i) each individual plaque studied contained viral DNA of predominantly one genotype only (90% or greater); (R) four genotypes in total (aBCD, aBCd, abcD, and abcd) were observed with high frequency; (id) four other theoretically possible genotypes (aBcD, aBcd, abCD, and abCd) were not observed in the 18 plaques studied; and (iv) each of Table 1. Genotypic analysis
Genotype
No. of plaques/genotype
aBCD aBCd abcD abcd
5 2 3 6*
* The original number of plaques with the abed genotype was eight. This value is corrected for the presence of plaques resulting from CR homoduplex formation (see Materials and Methods and text).
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Proc. Natl. Acad. Sci. USA 73 (1976)
Table 2. Analysis of marker segregation* No. of
No. of
Genotype
plaques
Genotype
plaques
aB ab aC ac aD ad BC Bc bC bc
7 9 7 9 8 8 7 0 0 9
CD Cd cD cd BD Bd bD bd
5 2 3 6 5 2 3 6
* The values presented in this table show the frequency of association of two markers of the genotypes of the 16 plaque isolates presented in Table 1.
the unselected markers (a has been selected) are observed with approximately equal frequency (D types equal d types, C types equal c types, and B types equal b types). In Table 2, the data of Table 1 have been rearranged to facilitate the association of any two markers. This presentation of results reveals that: (i) segregation of B and C or b and c markers is not observed (i.e., Bc and bC are not observed); (ii) all markers other than BC and bc segregate independently (e.g., aB, ab, aD, ad, Bd, BD, etc., all appear at high frequencies). The above analyses were performed on DNA derived from virus from the original heteroduplex DNA plaques that had been passaged at 38.50. Thus, any TS virus present in the friginal plaques would have been selected against. TS virus in the original plaques would have been expected to be carried by complementation with the non-TS virus present. Such non-TS virus could have been derived from replication prior to any repair or recombination during or immediately after DNA replication or simply by contamination. Virus from 12 of the original 16 heteroduplex-derived plaques were replaqued at 31.50. Virus from approximately 10 of these secondary plaques (derived from each of the 12 original plaque isolates) were tested for their temperature sensitivity. Of the 120 total plaques tested, only two were found to be temperature sensitive. One of these had a genotype of ABCd and was derived from an original plaque with an aBCD genotype, whereas the other had a genotype of ABCD and was derived from an original plaque with an abcd genotype. DISCUSSION Heteroduplex polyoma viral DNA was constructed; one strand of the heteroduplex DNA was derived from the TS-A variant, the other strand from the CR variant. Since the TS-A and CR parental variants differ genotypically at four sites distributed around the polyoma genome (Fig. 1), the heteroduplex DNA contained at least four mismatched base-pair regions. The progeny viral DNAs produced from infection of mouse cells with the heteroduplex DNA were analyzed genotypically. During progeny virus production, one region of the CR DNA strand was selected phenotypically. The other three genotypic differences were phenotypically silent mutations and were therefore genotypic markers with no apparent selective advantage. The silent genotypic markers were detected in the progeny viral DNA by restriction endonuclease analysis. The results of the genotypic analysis revealed that each individual TS-A/CR heteroduplex DNA infection produced nontemperature-sensitive progeny virus containing only one
genotype. Of the 16 individual heteroduplex infections studied, predominantly four genotypically distinct virus types were detected in total. Three of the four genotypes contained markers derived from both parental DNAs, and each of the four genotypes appeared in approximately equal proportion. To interpret these results, we have considered the effects of DNA replication, DNA recombination, and DNA repair on the segregation of genetic markers. DNA replication of the TS-A/CR heteroduplex would separate the TS-A and CR strands, resulting in two progeny viral genotypes: TS-A (ABCD) and CR (abed) (see Fig. 1). Due to the selection procedure used (ability to plaque at 38.50), plaques due to TS-A (ABCD) genotype alone would not be observed. Since 10 of the 16 plaques were genotypes containing a mixture of parental markers (aBCd, aBCD, and abeD), DNA replication alone cannot account for our results. Furthermore, our analyses revealed that only one of the six abed plaques contained a low level (approximately 10%) of virus of the ABCD genotype. Although this ABCD virus probably is a contaminant of the original TS-A (ABCD) DNA used to form the heteroduplexes, we cannot rule out that in a small number of cases DNA replication precedes the repair process. Recombination between the CR (abed) and TS-A (ABCD) DNAs immediately after DNA replication of the heteroduplex DNA could result in the observed mixing of the parental genotypic markers. Since over 60% of the individual infections produced progeny of a mixed parental genotype, the frequency of recombination would have to be at least 60% to explain our results. The recombination frequency of polyoma viruses was determined by genetic analysis to be 0.24% (25) and is therefore too low to explain our results. Such genetic analyses, however, provide no estimate of the recombination frequency between sister strands (intramolecular recombination during replication). Density label studies, which established the semiconservative nature of polyoma DNA replication, detected no evidence of frequent intermolecular or intramolecular recombination (26). It is therefore unlikely that recombination is responsible for the observed genotypic mixing. Nevertheless, we attempted to determine if intramolecular recombination contributed to the genotypic mixing observed in our experiments. Intramolecular recombination between the sister strands of a replicating heteroduplex molecule is expected to result in at least two nonparental genotypes in the progeny virus of individual heteroduplex infections. Since at least 60% of the replicating molecules must undergo recombination to explain our results by the recombination rationale, the two DNA molecules produced during recombination must be viable in order to achieve a reasonable amount of net synthesis of DNA during DNA replication. Of the plaques analyzed containing nonparental genotypes, a low level of virus of a second genotype was detected in only one plaque. The genotypes of the viruses present in this plaque (majority-aBCD, minority-ABCd) are not reciprocal recombinants, and since the order of DNA replication of the markers is Dd, Aa, Cc, Bb, it is unlikely that these genotypes were generated from a single recombination event. Most likely the ABCd genotype arose independently and is merely a contaminant in the aBCd virus plaque. An unequivocable interpretation of these results depends on a knowledge of the extent to which non-TS virus can complement a TS mutant during plaque formation at 38.5°. We cannot completely eliminate the possibility that reciprocal intramolecular recombination during replication contributes to the observed mixing of genotypic marker in the progeny virus. A simple explanation for our results is that cellular mechanisms are present which correct the mismatched base-pair regions in the heteroduplex before DNA replication, resulting in
Biochemistry: Miller et al. the appearance of a single genotype from each individual heteroduplex infection and the mixing of the parental genotypic markers. From the frequency of appearance of the various
Proc. Natl. Acad. Sci. USA 73 (1976)
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point is that mouse cells possess an extremely efficient correction mechanism which results in the correction of mismatched base-pair regions before the completion of polyoma virus DNA replication. Since polyoma has such a limited capacity for genetic coding of enzymes (5200 nucleotide base-pairs), we assume that it does not code for repair enzymes since this is a luxury only large viruses can afford. However, polyoma is able to induce several DNA metabolizing enzymes, and it is possible that cellular enzymes involved in excision repair are induced after polyoma infection.. The technique we used may prove to be a most valuable tool in studying gene conversion in mammalian cells. The technique will also be a useful method for transferring various genotypic markers from one virus strain to another. We thank Moira Griffiths for her excellent technical assistance, Brad Ozanne for helpful discussions on intramolecular recombination, and Beverly Griffin who supplied much of the information concerning the location of restriction endonuclease sites. L.K.M. was supported by a
genotypes it would appear that either sequence of the mismatched region is repaired with approximately equal probability. Our studies are limited to three markers, and the number of plaques analyzed is too low for an extensive statistical analysis. Two of the genotypic markers studied (B and C, see Fig. 1) may be point mutations generated by nitrous acid mutagenesis used for the original isolation of the parental variants (19). The third genotypic marker studied (D, see Fig. 1) is an addition of approximately 18 bases in one of the DNA strands of the heteroduplex DNA (23). The exact structure of this region in the heteroduplex molecule is not known, but the results indicate that there is no bias in the strand repaired in this region (see Table 2, aD equals ad). It would appear that the enzymes correcting a mismatched base-pair region show no preferential repair of DNA strands with larger single-stranded regions. Various addition/deletion sequences would have to be studied, Public Health Service Postdoctoral Fellowship (1F22 CA 01198-01) however, to determine if this lack of bias generally occurs. from the National Cancer Institute, U.S.A. Those genotypic markers that are separated by stretches of DNA approximately 600 nucleotides or more in length (see Fig. 1. Miller, L. K. & Fried, M. (1976) J. Virol. 18, in press. 2. Miller, L. K. & Fried, M. (1976) Nature 259,598-601. 2) segregate independently of each other (see Table 2). This 3. Spatz, H. Ch. & Trautner, T. A. (1970) Mol. Gen. Genet. 109, observation suggests that the number of nucleotides excised by 84-106. exonucleases and replaced by DNA polymerase during excision 4. Baas, P. D. & Janz, H. S. (1972) J. Mol. Biol. 63,557-568. repair does not usually exceed more than approximately 600 5. Wildenberg, J. & Meselson, M. (1975) Proc. Nat!. Acad. Sci. USA nucleotides. Two of the markers we studied always appeared 72,2202-2206. in the viruses together progeny (BC and bc, see Table 2). These 6. White, R. L. & Fox, M. S. (1975) Proc. Natl. Acad. Sci. USA 71, markers are separated by a length of approximately 90 nu1544-1548. cleotides. Since only one set of markers studied showed this 7. White, R. L. & Fox, M. S. (1975) Mol. Gen. Genet. 141, 163property, we cannot rule out that this result is due to some pe171. 8. Enea, V., Vovis, G. F. & Zinder, N. D. (1975) J. Mol. Biol. 96, culiarity in this region of the heteroduplex molecule. Since the 495-509. genotype of wild-type Pasadena large plaque DNA is aBcD (A2 9. Setlow, R. B. & Carrier, W. L. (1964) Proc. Natl. Acad. Sci. USA type) or aBcd (A3 type) (23), we know that the Bc genotype is 51,226-231. viable. The simplest explanation of the result would be corepair 10. Boyce, R. P. & Howard-Flanders, P. (1964) Proc. Nat!. Acad. Sci. of the mismatched regions. Such an interpretation is consistent USA 51, 293-300. with biochemical measurements of the length of DNA repaired 11. Cleaver, J. E. (1974) in Advances in Radiation Biology, eds. Lett, during excision repair in mammalian cells being approximately J. T., Adler, H. & Zelle, M. (Academic Press, New York), Vol. 4, 100 nucleotides (for a review, see ref. 11). The interpretation pp. 1-75. is also consistent with genetic evidence in lower eukaryotes of 12. Abrahams, P. J. & Van Der Eb, A. J. (1976) Mutat. Res. 35, coconversion of closely linked genetic markers (for a review, 13-22. see ref. 13), although some evidence in fungi suggests that repair 13. Holliday, R. (1974) Genetics 78, 273-387. 14. Hotchkiss, R. D. (1974) Genetics 78,247-257. may extend across approximately 700 nucleotides (17). Lai and 15. Whitehouse, H. L. K. (1974) Genet. Res. 24,,251-279. Nathans observe that genetic markers 250 nucleotides apart can 16. Carlson, P. S. (1971) Genet. Res. 17,53-81. segregate independently in monkey cells infected with simian 17. Hurst, D. D., Fogel, S. & Mortimer, R. K. (1972) Proc. Nat!. Acad. virus 40 (18), but the frequency of this occurring is difficult to Sci. USA 69, 101-105. estimate from the approach they used. 18. Lai, C-J., & Nathans, D. (1975) Virology 66,70-81. From the results presented here, we are not able to determine 19. Fried, M. (1965) Virology 25,669-671. whether there is a preferred strand for repair, since the het20. Hirt, B. (1967) J. Mol. Biol. 26,365-369. eroduplexes have been constructed to contain either the Watson 21. Fried, M. (1974) J. Virol. 13, 939-946. or Crick strands of both parental DNAs. Our results, however, 22. Radloff, R., Bauer, W. & Vinograd, J. (1967) Proc. Natl. Acad. Sci. USA 57, 1514-1521. suggest that repair can occur on both strands of the same DNA! 23. Fried, M., Griffin, B. E., Lund, E. & Robberson, D. L. (1974) Cold prior to DNA replication and probably proceeds to completion Spring Harbor Symp. Quant. Biol. 39,45-52. prior to replication in most cases. We assume that the correction 24. Griffin, B. E., Fried, M. & Cowie, A. (1974) Proc. Nat!. Acad. Sci. is, in part, stimulated by the presence of mismatched base-pair USA 71, 2077-2081. regions in the DNA. We note, however, that each heteroduplex 25. A. & Di Mayorca, G. (1971) Lepetit Co!!oq. Biol. Med. Ishikawa, DNA contained two single-strand disruptions upon infection 2,294-299. as well as other possible damage introduced during the purifi26. Hirt, B. (1966) Proc. Natl. Acad. Sci. USA 55,997-1004. cation of the DNA and construction of the heteroduplex. The 27. Griffin, B. E. & Fried, M. (1975) Nature 256, 175-179. x-irradiation technique used, for instance, probably introduced 28. Strniste, G. F., Armel, P. R. & Wallace, S. S. (1975) Radiat. Res. one alkali-labile bond per heteroduplex DNA (28). The essential 62,573.
Vol. 73, No. 9, pp. 3073-077, September 1976 Biochemistry
Fate of mismatched base-pair regions in polyoma heteroduplex DNA during infection of mouse cells (genotypic markers/restriction endonucleases/repair/recombination/replication)
Lois K. MILLER*, BEVERLEY E. COOKE, AND MIKE FRIED Imperial Cancer Research Fund, P.O. Box 123, Lincoln's Inn Fields, London WC2A 3PX, England
Communicated by Renato Dulbecco, June 16,1976
ABSTRACT Heteroduplex DNA has been constructed from two variants of polyoma virus that differ genotypically at four distinct sites. The genotypes of the progeny virus derived from infections of mouse cells with single heteroduplexes have been analyzed to determine how the genotypic markers of the parental heteroduplex segregate. Markers that are separated by a length of DNA greater than 600 nucleotides segregate independently. Segregation was not detected between two markers separated by only approximatel 90 nucleotides. We interpret these results on the basis of the correction of mismatched base-pair regions in the heteroduplex before the completion of DNA replication. We suggest that this technique provides valuable information concerning gene conversion in mammalian cells and permits the transfer of genotypic markers from one virus strain to another.
During experiments involving construction of a genetic map of the small, double-stranded DNA genome of polyoma virus (1, 2), we became interested in determining whether heteroduplex polyoma DNAs containing mismatched base-pairs are first replicated, resulting in the independent segregation of two DNAs of distinct genotypes, or whether other cellular mechanisms correct the mismatched base-pair region before the completion of DNA replication, resulting in DNA of one genotype only. The question was generally interesting with regard to DNA transitions in mammalian cells. The question was also important with respect to viral genetics in predicting the genotype of progeny virus produced from heteroduplex DNA infections in genetic mapping experiments involving the marker rescue technique (Hewick, Waterfield, Miller, and Fried, manuscript in preparation). Correction of mismatched base-pairs in heteroduplex DNA has been studied extensively in bacteria using heteroduplex phage DNA (3-8). These studies demonstrated that cellular mechanisms are present in prokaryotes that can correct (repair) mismatched base-pair regions in heteroduplex DNA before the completion of DNA replication. In OX174, the correction of a mismatched base-pair before replication depended on the position of the mismatched base-pair relative to the origin of DNA replication (4); the closer the mismatched base was to the origin, the less frequently it was corrected before DNA replication. Correction of mismatched base-pairs may involve four basic steps, similar to those described for the repair of UV-damaged DNA (9, 10). First, the mismatched base-pair may be recognized and marked by an endonucleolytic nick (single-strand break) in one of the two DNA strands. Second, the nucleotide Abbreviations: FI DNA, supercoiled circular DNA; FII DNA, relaxed (nicked) circular DNA, i.e., DNA containing a single-strand break; FEI DNA, linear DNA; EcoRI, restriction endonuclease RI from Escherichia coil; HhaI, HaeIII, HindIII, and HpaII, restriction endonucleases from Haemophilus haemolyticus, H. aegyptius, H. influenzae, and H. parainfluenzae, respectively; TS, temperature sensitive. *
Present address: Department of Bacteriology and Biochemistry, The University of Idaho, Moscow, Idaho 83843. 3073
sequences around this nick on the DNA strand may be degraded by exonuclease action, forming a single-stranded gap. This can be followed by DNA synthesis, which fills the gap by the action of DNA polymerase using the opposite strand sequences as a template. Finally, the end of a newly synthesized sequence can be joined to preexisting sequences on the repaired strand by the action of DNA ligase. Although it is known that mammalian cells possess an excision-repair system for correction of UV-damaged DNA (11, 12), information concerning the ability of mammalian cells to correct mismatched base-pairs is lacking. Studies of gene conversion and postmeiotic segregation in fungi and Drosophila suggest the formation of segments of heteroduplex DNA. As
an intermediate step in the recombination process with mismatched base-pair regions within the heteroduplex DNA segment corrected (13-17). -Mammalian systems may also contain enzymes for correction of mismatched base-pair regions. The results of Lai and Nathans (18) using heteroduplex DNA of simian virus have been explained on the basis of correction of mismatched base pairs in African green monkey cells. In order to answer the question of whether mismatched base-pairs of heteroduplex DNA were corrected before DNA replication occurred, we used a unique set of silent mutations of polyoma virus. These silent mutations are detectable by restriction endonuclease analysis, but they have no effect on the phenotype of the viruses. Each strand of the viral heteroduplex DNAs constructed contained four distinguishable genotypic markers, and the segregation of these markers in the progeny viruses could be monitored. MATERIALS AND METHODS Viruses and Preparation of Viral DNAs. The TS (temperature sensitive)-A variant (19) and the CR variant (Fried, unpublished results) were isolated from the Pasadena large plaque strain of polyoma virus. Virus stocks were derived from single plaque isolates and passaged at low multiplicity. Supercoiled viral DNA was purified from Hirt (20) supernatants of infected mouse embryo cells by CsCl/ethidium bromide equilibrium density centrifugation followed by sedimentation in neutral sucrose gradients C21). Heteroduplex Formation. The TS-A F1 DNA (supercoiled circular DNA) was randomly nicked by x-irradiation so that 30% FI was converted to FII (nicked circular DNA). The singly-nicked FII DNA was purified from the remaining FI by CsCl/ethidium bromide equilibrium density centrifugation (22). The CR FI DNA was cleaved into two fragments by incubation with HindIll restriction nuclease (from Haemophilus influenzae) at 370 in 10 mM Tris-HCl (pH 7.4)-6 mM MgCI2-1 mM dithiothreitol-50 mM NaCl. The enzyme was heat-denatured by incubation at 80° for 15 min. The TS-A FII DNA (1.1 ,ug/ml) and HindIII-cleaved CR DNA (11 ,g/ml) were denatured in 0.4 M NaOH at room temperature for 20 min. The
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Hindill
Hhai 0 "-
Infect cells at 390C The "a" Phenotype is selected
Analyze Genotype of Progeny from Single Heteroduplex
FIG. 1. The experimental design. Two variants of polyoma, TS-A and CR, differ genotypically at four distinct sites (A versus a, B versus b, C versus c, and D versus d). The approximate position of genotypically different sites (0, *, X, *) are marked relative to the origin of bidirectional, semiconservative DNA replication (OR). Heteroduplexes between TS-A and CR DNAs are formed by denaturation and reannealing of nicked parental DNAs (see Materials and Methods). Secondary whole mouse embryo cells are infected with heteroduplex DNAs. Plaques are developed at 38.50 to select for the "a" phenotype. The DNAs derived from individual plaques are analyzed for the genotype markers Bb, Cc, and Dd by digestion with restriction nucleases (see Materials and Methods).
solution was neutralized with HCl and buffered by 0.16 M sodium phosphate buffer (pH 7.0) and the strands were reannealed at 68° for 30 min. The DNA solution was diluted with Dulbecco's phosphate-buffered saline solution minus Mg++ and Ca++. Infection of Cells with Heteroduplex DNA. DNA was spread on mouse embryo cells and the cells were incubated (1). After 8 days at 38.50 the cells were stained with nbutral red and plaques picked the following day. Virus stocks were prepared at 38.50 from each individual plaque. Approximately 1 X 104 plaque-forming units/.ug of heteroduplex DNA appeared at 38.50. Less than 1 X 102 plaque-forming units/Ag appeared on plates containing TS-A without CR HindIll-digested DNA. The CR HindIII fragments alone produced plaques at one-ninth the frequency of CR HindIII fragments with TS-A FII in heteroduplex form described above. Genotypic Analysis with Restriction Nucleases. Supercoiled FI DNA was prepared as described above from infection of whole mouse embryo cells with virus stocks derived from single plaques. The polyoma FI DNAs were digested with endonuclease HpaII (from H. parainfluenzae), HhaI (from H. haemolyticus) or HaeIII (from H. aegyptius) in 10 mM TrisHCl (pH 7.5)-10 mM MgCl2-1 mM dithiothreitol. For each enzyme digestion, approximately 2 Mg of FI DNA were digested at 370 for approximately 3 hr with sufficient enzyme for complete digestion. The DNA fragments were then analyzed on 20 X 20 cm slab gels. For DNA digested with HhaI and HaeIII, 1.4% agarose slab gels were used. For HpaII-digested DNA, 3.3% acrylamide-0.17% bisacrylamide-0.5% agarose gels
FIG. 2. The physical map of polyoma DNA indicating the location of the EcoRI, HinHI, HhaI, and HpaII restriction endonuclease cleavage sites, the origin (0) and termination of DNA synthesis (adapted from ref. 14, and Griffin, personal communication). The circular polyoma DNA genome is composed of approximately 5200 nucleotide pairs. The map is divided into 100 map units with the EcoRI cleavage site at 00 map units. The TS-A is cleaved by HhaI at the three sites indicated; the CR variant lacks the HhaI cleavage site at 26.4 map units (denoted as B or b in Fig. 1). HaeIll cleaves CR approximately 24 times. Only one of these cleavage sites is denoted on the map (HaeIII*) at 28.4 map units. The TS-A variant lacks this HaeIII site (denoted as C or c in Fig. 1).
were used. The restriction nuclease fragment patterns were visualized by ethidium bromide staining.
RESULTS Differences Between the TS-A and CR Variants. The experimental design for studying the repair of polyoma heteroduplex DNA is schematically presented in Fig. 1. Heteroduplexes were formed between the two polyoma variants, TS-A and CR, which differ genotypically in four distinct sites on the genome. One site is a mutation that governs the ability of the variant strains to produce infectious virus at 38.50. The CR strain will form plaques at 38.50, whereas the TS-A strain will not. The position of the mutation responsible for this temperature-sensitive (TS) property of TS-A has been located by a marker rescue technique (1) and is located between the Hindfll restriction enzyme cleavage site at 1.0 map unit and the Hha I restriction enzyme cleavage site at 14.0 map units on the polyoma physical map (see Fig. 2). This site has been designated as A for TS-A and "a" for CR (see Fig. 1). A second site where the two variants differ is at the HhaI site at 26.4 map units on the polyoma physical map (Fig. 2). The TS-A variant contains the Hha cleavage site, whereas the CR variant does not (C. Barry and M. Griffiths, personal communication). Thus, when TS-A DNA is digested with HhaI, three fragments (46, 42, and 12%) are generated (27), whereas HhaI digestion of CR DNA results in the generation of only two fragments (58 and 42% of the genome) (see Fig. 3). This site at 26.4 map units (Griffin, personal communication) has been designated B for TS-A and b for CR (Fig. 1). The two variants also differ at another restriction enzyme cleavage site. This is at the HaeIII site at 28.2 map units on the physical map (Fig. 2; Griffin, personal communication) joining HaeIII fragment 6 and HaeIII fragment 20. The TS-A DNA, lacks this restriction enzyme site, resulting in the absence of these two fragments and the appearance of a new fragment (see Fig. 4). The new fragment migrates in gels to a position expected for a molecule of molecular weight equal to the com-
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B
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TS-A A2
C
u_ ...
do
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FIG. 3. HhaI restriction endonuclease fragment patterns of TS-A and CR DNAs. FI DNA of TS-A and CR DNA were digested with HhaI restriction endonuclease and the fragments were electrophoresed through 1.4% agarose gels in the presence of ethidium bromide. Photographs of the gels were taken under ultraviolet illumination; the negative print is shown above. (A) Control FIT, FIII (linear), and FT of undigested A2 DNA. (B) HhaI-digested TS-A DNA. (C) HhaT-digested CR DNA; arrows on right point to the TS-A fragments missing in the CR fragment pattern. Note the new fragment in the CR pattern, which is a combination of the two TS-A fragments.
bined molecular weight of HaeIll-6 and HaeTIT-20 fragments. This HaeIII site is designated C in TS-A and c in CR (Fig. 1).
The fourth site where the two variants differ is located in HpaTT-5, near the origin of DNA synthesis. HpaII-5 differs in size between different isolates of the Pasadena large plaque strain by approximately 0.3% of the genome or 18 base pairs (23). This difference is located between 70.7 and 72.7 map units of the polyoma physical map (Griffin, personal communication) (Fig. 2) and can easily be detected by the electrophoretic mobility of HpaII-5 in polyacrylamide gels (23}. The TS-A strain contains the more slowly migrating, or larger, Hpall-5. This site is designated D for TS-A and d for CR (Fig. 1). Formation of Heteroduplex DNA. Since the DNA strands of polyoma are difficult to separate, the method of heteroduplex formation was carefully designed and selection was applied to allow us to study only the heteroduplexes rather than the homoduplexes. To reduce the amount of infectious CR homoduplexes, we cleaved the CR FT DNA with HindIII, which cleaves the DNA twice: one cut is at 1.0 map unit, the other is at 45.0 map units (24). This not only permitted separation of strands under alkaline conditions, but also reduced the formation of infectious CR homoduplex DNA. To select against the TS-A homoduplexes, we isolated the virus analyzed from plaques formed at 38.5°, a temperature at which the TS-A homoduplexes will not form plaques. Thus, all the virus isolates studied, being selected at 38.5°, have the "a" genotype (see Tables 1 and 2). To favor heteroduplex formation, a 10-fold molar excess of CR HindITI fragments were added to the TS-A FIT DNA. After denaturation and renaturation (see Materials and Methods) the majority of the circular DNA molecules are heteroduplexes composed of one CR DNA strand with two nicks at HindITT sites and one TS-A DNA strand with one random nick in half of the heteroduplexes.. Under the conditions used the number of plaques appearing due to the
FIG. 4. The HaellI restriction nuclease fragment patterns of TS-A and A2 DNAs. 32P-Labeled FI DNA was prepared from 3T6 cells infected with TS-A or A2 viruses (24). Fl DNAs were digested with HaeIII (see Materials and Methods) and the fragments were electrophoresed through 7% polyacrylamide gels (20 X 40 cm). Autoradiographs of the resulting fragment patterns of the two DNAs are shown above. The arrow on the left points to the new fragment of TS-A, which is a combination of two smaller fragments seen in the A2 pattern marked by arrows on the right. With respect to these fragments, CR DNA has a similar HaeIII cutting pattern as A2 DNA.
reannealing of CR HindHI fragments is one-ninth the amount of plaques developed for CR/TS-A heteroduplexes. Since we know the genotype of the CR HindIII homoduplexes is abed, we can subtract out one abcd genotype for every nine plaques studied and thus correct for the contaminating level of CR homoduplex (Table 1). A diagrammatic representation of the heteroduplex formation with the location of the genotypic markers is shown in Fig. 1. Genotypic Analysis. Viral DNAs derived from 18 plaque isolates of heteroduplex DNA were analyzed by restriction enzymes (see Materials and Methods) and classified according to genotype (Table 1). There are four important observations to note from these results: (i) each individual plaque studied contained viral DNA of predominantly one genotype only (90% or greater); (R) four genotypes in total (aBCD, aBCd, abcD, and abcd) were observed with high frequency; (id) four other theoretically possible genotypes (aBcD, aBcd, abCD, and abCd) were not observed in the 18 plaques studied; and (iv) each of Table 1. Genotypic analysis
Genotype
No. of plaques/genotype
aBCD aBCd abcD abcd
5 2 3 6*
* The original number of plaques with the abed genotype was eight. This value is corrected for the presence of plaques resulting from CR homoduplex formation (see Materials and Methods and text).
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Table 2. Analysis of marker segregation* No. of
No. of
Genotype
plaques
Genotype
plaques
aB ab aC ac aD ad BC Bc bC bc
7 9 7 9 8 8 7 0 0 9
CD Cd cD cd BD Bd bD bd
5 2 3 6 5 2 3 6
* The values presented in this table show the frequency of association of two markers of the genotypes of the 16 plaque isolates presented in Table 1.
the unselected markers (a has been selected) are observed with approximately equal frequency (D types equal d types, C types equal c types, and B types equal b types). In Table 2, the data of Table 1 have been rearranged to facilitate the association of any two markers. This presentation of results reveals that: (i) segregation of B and C or b and c markers is not observed (i.e., Bc and bC are not observed); (ii) all markers other than BC and bc segregate independently (e.g., aB, ab, aD, ad, Bd, BD, etc., all appear at high frequencies). The above analyses were performed on DNA derived from virus from the original heteroduplex DNA plaques that had been passaged at 38.50. Thus, any TS virus present in the friginal plaques would have been selected against. TS virus in the original plaques would have been expected to be carried by complementation with the non-TS virus present. Such non-TS virus could have been derived from replication prior to any repair or recombination during or immediately after DNA replication or simply by contamination. Virus from 12 of the original 16 heteroduplex-derived plaques were replaqued at 31.50. Virus from approximately 10 of these secondary plaques (derived from each of the 12 original plaque isolates) were tested for their temperature sensitivity. Of the 120 total plaques tested, only two were found to be temperature sensitive. One of these had a genotype of ABCd and was derived from an original plaque with an aBCD genotype, whereas the other had a genotype of ABCD and was derived from an original plaque with an abcd genotype. DISCUSSION Heteroduplex polyoma viral DNA was constructed; one strand of the heteroduplex DNA was derived from the TS-A variant, the other strand from the CR variant. Since the TS-A and CR parental variants differ genotypically at four sites distributed around the polyoma genome (Fig. 1), the heteroduplex DNA contained at least four mismatched base-pair regions. The progeny viral DNAs produced from infection of mouse cells with the heteroduplex DNA were analyzed genotypically. During progeny virus production, one region of the CR DNA strand was selected phenotypically. The other three genotypic differences were phenotypically silent mutations and were therefore genotypic markers with no apparent selective advantage. The silent genotypic markers were detected in the progeny viral DNA by restriction endonuclease analysis. The results of the genotypic analysis revealed that each individual TS-A/CR heteroduplex DNA infection produced nontemperature-sensitive progeny virus containing only one
genotype. Of the 16 individual heteroduplex infections studied, predominantly four genotypically distinct virus types were detected in total. Three of the four genotypes contained markers derived from both parental DNAs, and each of the four genotypes appeared in approximately equal proportion. To interpret these results, we have considered the effects of DNA replication, DNA recombination, and DNA repair on the segregation of genetic markers. DNA replication of the TS-A/CR heteroduplex would separate the TS-A and CR strands, resulting in two progeny viral genotypes: TS-A (ABCD) and CR (abed) (see Fig. 1). Due to the selection procedure used (ability to plaque at 38.50), plaques due to TS-A (ABCD) genotype alone would not be observed. Since 10 of the 16 plaques were genotypes containing a mixture of parental markers (aBCd, aBCD, and abeD), DNA replication alone cannot account for our results. Furthermore, our analyses revealed that only one of the six abed plaques contained a low level (approximately 10%) of virus of the ABCD genotype. Although this ABCD virus probably is a contaminant of the original TS-A (ABCD) DNA used to form the heteroduplexes, we cannot rule out that in a small number of cases DNA replication precedes the repair process. Recombination between the CR (abed) and TS-A (ABCD) DNAs immediately after DNA replication of the heteroduplex DNA could result in the observed mixing of the parental genotypic markers. Since over 60% of the individual infections produced progeny of a mixed parental genotype, the frequency of recombination would have to be at least 60% to explain our results. The recombination frequency of polyoma viruses was determined by genetic analysis to be 0.24% (25) and is therefore too low to explain our results. Such genetic analyses, however, provide no estimate of the recombination frequency between sister strands (intramolecular recombination during replication). Density label studies, which established the semiconservative nature of polyoma DNA replication, detected no evidence of frequent intermolecular or intramolecular recombination (26). It is therefore unlikely that recombination is responsible for the observed genotypic mixing. Nevertheless, we attempted to determine if intramolecular recombination contributed to the genotypic mixing observed in our experiments. Intramolecular recombination between the sister strands of a replicating heteroduplex molecule is expected to result in at least two nonparental genotypes in the progeny virus of individual heteroduplex infections. Since at least 60% of the replicating molecules must undergo recombination to explain our results by the recombination rationale, the two DNA molecules produced during recombination must be viable in order to achieve a reasonable amount of net synthesis of DNA during DNA replication. Of the plaques analyzed containing nonparental genotypes, a low level of virus of a second genotype was detected in only one plaque. The genotypes of the viruses present in this plaque (majority-aBCD, minority-ABCd) are not reciprocal recombinants, and since the order of DNA replication of the markers is Dd, Aa, Cc, Bb, it is unlikely that these genotypes were generated from a single recombination event. Most likely the ABCd genotype arose independently and is merely a contaminant in the aBCd virus plaque. An unequivocable interpretation of these results depends on a knowledge of the extent to which non-TS virus can complement a TS mutant during plaque formation at 38.5°. We cannot completely eliminate the possibility that reciprocal intramolecular recombination during replication contributes to the observed mixing of genotypic marker in the progeny virus. A simple explanation for our results is that cellular mechanisms are present which correct the mismatched base-pair regions in the heteroduplex before DNA replication, resulting in
Biochemistry: Miller et al. the appearance of a single genotype from each individual heteroduplex infection and the mixing of the parental genotypic markers. From the frequency of appearance of the various
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point is that mouse cells possess an extremely efficient correction mechanism which results in the correction of mismatched base-pair regions before the completion of polyoma virus DNA replication. Since polyoma has such a limited capacity for genetic coding of enzymes (5200 nucleotide base-pairs), we assume that it does not code for repair enzymes since this is a luxury only large viruses can afford. However, polyoma is able to induce several DNA metabolizing enzymes, and it is possible that cellular enzymes involved in excision repair are induced after polyoma infection.. The technique we used may prove to be a most valuable tool in studying gene conversion in mammalian cells. The technique will also be a useful method for transferring various genotypic markers from one virus strain to another. We thank Moira Griffiths for her excellent technical assistance, Brad Ozanne for helpful discussions on intramolecular recombination, and Beverly Griffin who supplied much of the information concerning the location of restriction endonuclease sites. L.K.M. was supported by a
genotypes it would appear that either sequence of the mismatched region is repaired with approximately equal probability. Our studies are limited to three markers, and the number of plaques analyzed is too low for an extensive statistical analysis. Two of the genotypic markers studied (B and C, see Fig. 1) may be point mutations generated by nitrous acid mutagenesis used for the original isolation of the parental variants (19). The third genotypic marker studied (D, see Fig. 1) is an addition of approximately 18 bases in one of the DNA strands of the heteroduplex DNA (23). The exact structure of this region in the heteroduplex molecule is not known, but the results indicate that there is no bias in the strand repaired in this region (see Table 2, aD equals ad). It would appear that the enzymes correcting a mismatched base-pair region show no preferential repair of DNA strands with larger single-stranded regions. Various addition/deletion sequences would have to be studied, Public Health Service Postdoctoral Fellowship (1F22 CA 01198-01) however, to determine if this lack of bias generally occurs. from the National Cancer Institute, U.S.A. Those genotypic markers that are separated by stretches of DNA approximately 600 nucleotides or more in length (see Fig. 1. Miller, L. K. & Fried, M. (1976) J. Virol. 18, in press. 2. Miller, L. K. & Fried, M. (1976) Nature 259,598-601. 2) segregate independently of each other (see Table 2). This 3. Spatz, H. Ch. & Trautner, T. A. (1970) Mol. Gen. Genet. 109, observation suggests that the number of nucleotides excised by 84-106. exonucleases and replaced by DNA polymerase during excision 4. Baas, P. D. & Janz, H. S. (1972) J. Mol. Biol. 63,557-568. repair does not usually exceed more than approximately 600 5. Wildenberg, J. & Meselson, M. (1975) Proc. Nat!. Acad. Sci. USA nucleotides. Two of the markers we studied always appeared 72,2202-2206. in the viruses together progeny (BC and bc, see Table 2). These 6. White, R. L. & Fox, M. S. (1975) Proc. Natl. Acad. Sci. USA 71, markers are separated by a length of approximately 90 nu1544-1548. cleotides. Since only one set of markers studied showed this 7. White, R. L. & Fox, M. S. (1975) Mol. Gen. Genet. 141, 163property, we cannot rule out that this result is due to some pe171. 8. Enea, V., Vovis, G. F. & Zinder, N. D. (1975) J. Mol. Biol. 96, culiarity in this region of the heteroduplex molecule. Since the 495-509. genotype of wild-type Pasadena large plaque DNA is aBcD (A2 9. Setlow, R. B. & Carrier, W. L. (1964) Proc. Natl. Acad. Sci. USA type) or aBcd (A3 type) (23), we know that the Bc genotype is 51,226-231. viable. The simplest explanation of the result would be corepair 10. Boyce, R. P. & Howard-Flanders, P. (1964) Proc. Nat!. Acad. Sci. of the mismatched regions. Such an interpretation is consistent USA 51, 293-300. with biochemical measurements of the length of DNA repaired 11. Cleaver, J. E. (1974) in Advances in Radiation Biology, eds. Lett, during excision repair in mammalian cells being approximately J. T., Adler, H. & Zelle, M. (Academic Press, New York), Vol. 4, 100 nucleotides (for a review, see ref. 11). The interpretation pp. 1-75. is also consistent with genetic evidence in lower eukaryotes of 12. Abrahams, P. J. & Van Der Eb, A. J. (1976) Mutat. Res. 35, coconversion of closely linked genetic markers (for a review, 13-22. see ref. 13), although some evidence in fungi suggests that repair 13. Holliday, R. (1974) Genetics 78, 273-387. 14. Hotchkiss, R. D. (1974) Genetics 78,247-257. may extend across approximately 700 nucleotides (17). Lai and 15. Whitehouse, H. L. K. (1974) Genet. Res. 24,,251-279. Nathans observe that genetic markers 250 nucleotides apart can 16. Carlson, P. S. (1971) Genet. Res. 17,53-81. segregate independently in monkey cells infected with simian 17. Hurst, D. D., Fogel, S. & Mortimer, R. K. (1972) Proc. Nat!. Acad. virus 40 (18), but the frequency of this occurring is difficult to Sci. USA 69, 101-105. estimate from the approach they used. 18. Lai, C-J., & Nathans, D. (1975) Virology 66,70-81. From the results presented here, we are not able to determine 19. Fried, M. (1965) Virology 25,669-671. whether there is a preferred strand for repair, since the het20. Hirt, B. (1967) J. Mol. Biol. 26,365-369. eroduplexes have been constructed to contain either the Watson 21. Fried, M. (1974) J. Virol. 13, 939-946. or Crick strands of both parental DNAs. Our results, however, 22. Radloff, R., Bauer, W. & Vinograd, J. (1967) Proc. Natl. Acad. Sci. USA 57, 1514-1521. suggest that repair can occur on both strands of the same DNA! 23. Fried, M., Griffin, B. E., Lund, E. & Robberson, D. L. (1974) Cold prior to DNA replication and probably proceeds to completion Spring Harbor Symp. Quant. Biol. 39,45-52. prior to replication in most cases. We assume that the correction 24. Griffin, B. E., Fried, M. & Cowie, A. (1974) Proc. Nat!. Acad. Sci. is, in part, stimulated by the presence of mismatched base-pair USA 71, 2077-2081. regions in the DNA. We note, however, that each heteroduplex 25. A. & Di Mayorca, G. (1971) Lepetit Co!!oq. Biol. Med. Ishikawa, DNA contained two single-strand disruptions upon infection 2,294-299. as well as other possible damage introduced during the purifi26. Hirt, B. (1966) Proc. Natl. Acad. Sci. USA 55,997-1004. cation of the DNA and construction of the heteroduplex. The 27. Griffin, B. E. & Fried, M. (1975) Nature 256, 175-179. x-irradiation technique used, for instance, probably introduced 28. Strniste, G. F., Armel, P. R. & Wallace, S. S. (1975) Radiat. Res. one alkali-labile bond per heteroduplex DNA (28). The essential 62,573.
