JOURNAL OF VIROLOGY, Nov. 1976, p. 441-445
Vol. 20, No. 2 Printed in U.S.A.
Copyright C 1976 American Society for Microbiology
Evidence That Mismatched Bases in Heteroduplex T4 Bacteriophage Are Recognized In Vivo' HILLARD BERGER2 AND DREW PARDOLL3 Department ofBiology, The Johns Hopkins University, Baltimore, Maryland 21218 Received for publication 1 June 1976
T4 heteroduplex heterozygotes are lost selectively after prolonged incubation of phage-infected Escherichia coli cells under nonreplicating conditions. The loss of heterozygosity occurs for four out of six ril sites tested and is not dependent upon T4 v gene function. The results are interpreted to indicate the existence of a base-specific system for the recognition of mismatched bases in intracellular DNA.
Many models for genetic recombination postulate the existence of a mechanism for the repair of mismatched bases in heteroduplex DNA. Genetic phenomena which may be explained by mechanisms for "prereplicative restoration" of complementarity in heteroduplex DNA are: (i) gene conversion in fission fungi and Ascobulus immersus (see reference 16 for a review), (ii) high negative interference observed in multifactor phage crosses (reviewed in reference 10), (iii) short distance marker effects in phage and bacterial crosses (11, 18), and (iv) low integration efficiency in transformation (3, 8). Genetic and physicochemical experiments have established that heteroduplex DNA is an intermediate in recombination (reviewed in reference 10). Recent experiments with Bacillus subtilis phage SPP 1 (15), Escherichia coli phage X (19), and Pneumococcus (12) indicate that the DNA in these systems is subject to mismatch correction; however, data indicating that heteroduplex DNA is subject to site-specific repair is lacking in the bacteriophage T4 system. To investigate this point we have singly infected cells with T4 phage populations enriched for heteroduplex heterozygotes (13) and examined these infected cells for loss of segregating capacity (mottled plaques) under nonreplicating conditions. The results indicate: (i) after prolonged incubation under nonreplicating conditions, the capacity of heterozygous T4 phage to segregate is reduced, (ii) this apparent recognition of heterozygous DNA is marker-specific, (iii) the apparent repair is not dependent on the ' Address reprint requests to: Chairman, Department of Biology, The Johns Hopkins University, Baltimore, MD 21218. 2 Deceased 28 October 1975. 3 Present address: The Johns Hopkins School of Medicine, Baltimore, MD 21205.
v gene-encoded endonuclease V (5), and (iv) the process is partially dependent upon phage-specific protein synthesis. MATERIALS AND METHODS Bacteria. E. coli B was used as the host for all crosses and assays, except for determination of reversions to rIl+, in which case K-12(X) was used as indicator. Phage mutants. The rH mutants used have been previously described and are derived from T4B (2, 17). T4D tsG40 (gene 431) was repeatedly backcrossed to T4B and used to make double mutants. The v gene mutant was originally obtained from W. Harm (6) and was extensively backcrossed to T4B before use in the construction of the v-rIl-ts and v-ts mutants. Preparation of phage stocks enriched in heterozygotes. Phage stocks containing 4 to 5% heterozygotes for each of six different rHI sites were prepared by crossing tsG40-rII phage with tsG40-rII+ phage under conditions in which DNA synthesis was inhibited by addition of 5-fluorodeoxyuridine (FUdR) (13). Crosses were done at 30'C, and the plates were incubated from 12 to 16 h at 32'C before scoring for mottled plaques. All scoring was from coded plates to prevent bias. Holding experiments. Phage stocks enriched for heterozygotes were used to infect E. coli B at 3 x 108 cells/ml, with a multiplicity of infection of 0.01. Infection was done in M9+ medium supplemented with FUdR (20 ug/ml), uracil (40 Ag/ml), and Ltryptophan (20 ,ug/ml) at 42°C. Under these conditions there was no measurable DNA replication (H. Berger, unpublished data). At 5 min after infection the culture was diluted 50-fold into the same medium containing anti-T4 serum. The antiserum was added to inactivate unadsorbed phage and to inactivate any rare phage released during the holding period. The antiserum coupled with the dilution from the initial adsorption mixture insured that only primary infective centers would be scored upon plating after the hold. At various times the hold was terminated by dilution and plating at 32°C. The frequency of infected cells producing mottled 441
442
J. VIROL.
BERGER AND PARDOLL
plaques was determined after 12 to 15 h of incubation from coded plates. When chloramphenicol was used it was added 3 min after infection to a final concentration of 250 ,ug/ml. Approximately 50 mottled plaques were scored for each determination. The average percentage of mottled plaques was based on at least five and as many as eight individual trials. Media. The media, preparation of indicator bacteria, and standard cross procedures to prepare multiple mutants are as described by Krisch et al. (7).
RESULTS The rlI mutants used in this study have been characterized by their response to specific mutagens (2, 17). A summary of tentative base pair assignments and type of mutation is shown in Table 1. Also included are the results of the reversion response to 5-bromodeoxyuridine performed in conjunction with the present study. These results confirm the previous findings, with the exception of rSM94, which was previously classified as a transversion mutation but which appears to be a transition change. As described in Materials and Methods, each of the rII-tsG40 double mutants was crossed with tsG40 in the presence of FUdR to produce a phage population enriched for heterozygotes. Table 2 shows the average percentage of heterozygotes (mottled plaques) and yield obtained from these crosses. Since all of the phage contained the tsG40 mutation, plates were incubated at 32°C before mottled plaques were scored. The heterozygote-enriched populations
TABLE 2. Preparation of phage stocks enriched for heterozygotes Cross Cross
rSN103-tsG40 x tsG40 rC204-tsG40 x tsG40 rNT88-tsG40 x tsG40 rUV74-tsG40 x tsG40 rEM84-tsG40 x tsG40 rSM94-tsG40 x tsG40
Burst size Mottled(%) fected (phage/inbacteplaques rium)
4.8 5.0 4.3 5.4 4.0 4.3
1.1 1.8 0.7 0.8 2.1 2.0
were then used to infect E. coli B in the presence of FUdR at the nonpermissive temperature. The infected cells were held under these nonreplicating conditions for various times, plated under permissive conditions, and scored for the frequency of infected cells capable of rIl segregation as measured by the appearance of mottled plaques. The data are shown in Table 3 and graphically depicted in Fig. 1. For four of the six sites (mutants rSN103, rC204, rNT88, and rUV74) there is a time-dependent decrease in frequency of infective centers containing segregants, significant at P = 0.05 for late sampling times (9). Possible explanations for the time-dependent loss in segregating capacity are: (i) the operation of a site-specific repair process; (ii) a generalized inactivation of DNA strands; (iii) random repair similar to the process described by Freese and Freese (4) to explain UV-stimulated production of pure clones after hydroxylamine treatment of T4 phage; (iv) incomplete genomic TABLE 1. Base substitutions in rII mutants sampling attributable to decreased burst sizes resulting from the maintenance of infected cells Response under nonreplicating conditions for extended to Base Misperiods. Each of the latter three explanations BUdR match Mutant Most likely pair at Referpredicts a decline in heterozygosity for all rIl mutant type mutant ence (muta- repairc markers. In contrast, as shown in Table 3 and gen/ site a spontain Fig. 1, the frequency of segregating infective neous)" centers remains nearly constant for the rSM94 + 56 2 and rEM84 mutations. We have also measured rSN103 Transition G*C 7 2 rEM84 Transition A-T the effect of the FUdR holding conditions on + 2 2 rC204 Transition A-T average burst size (Table 4). Although pro+ 56 2 rNT88 Transition G * C longed maintenance of infected cells under non+ 3 18 rUV74d Transver- A-T replicating conditions does decrease phage sion? yields, the average phage production per pro65 18 rSM94d Transition G C ductive infective center is sufficiently large to a Abbreviations: G- C, Guanine -cytosine, A-T, suggest that all genomic types would be inadenine-thymine. cluded in an individual yield. To further show b Revertants were measured by plating on B and that mixed yields are efficiently recognized unK12 (X) after one cycle of growth in the presence of der the FUdR holding conditions, we have car100 ,ug of BUdR per ml in M9+ medium. set of reconstruction experiments. Data of this paper; +, present; -, none detected. ried out aFUdR holding conditions, cells were Using the d These mutations previously were classified as transversions. Mutation rSM94 responds as a tran- infected with an average multiplicity of infecsition mutation, whereas rUV74 gives a questiona- tion of 0.1 of tsG40 and tsG40-rUV74 phage and the frequency of mottled plaques, which arise ble response in our tests. c
443
RECOGNITION OF MISMATCHED BASES
VOL. 20, 1976 TABiE 3. Effects ofprolonged incubation without DNA synthesis on the segregation capacity of heteroduplex heterozygotes for six rI point-mutation sites Significantly
Hold-
Avg percent- N f ing age of mottled Marker time plaques (% ± trials (n) SE) (min)
ta
different from "010" trialb (P
=
0.05)
5.04 + 0.36 5 4.62 ± 0.11 5 1.05 No 3.84 ± 0.22 5 2.77 Yes 1.77 No 4.10 ± 0.39 5 3.80 ± 0.27 5 3.81 Yes 4.96 ± 0.25 5 rC204 4.34 ± 0.08 5 2.34 Yes 3.60 ± 0.08 5 5.17 Yes 3.58 + 0.24 5 3.50 Yes 3.40 ± 0.23 5 4.56 Yes 4.03 ± 0.40 8 rNT88 3.45 ± 0.29 8 1.17 No 3.28 + 0.22 8 1.64 No 2.80 + 0.21 8 2.70 Yes 2 54 0.26 8 3.10 Yes 5.30 ± 0.30 7 rUV74 4.90 + 0.37 7 0.83 No 50 4.39 + 0.39 7 1.84 Yes 70 3.94 ± 0.29 7 3.16 Yes 90 3.78 ± 0.24 7 4.83 Yes rEM84 0-10 3.54 ± 0.40 5 30 3.70 ± 0.40 5 0.48 No 50 3.28 ± 0.37 5 0.48 No 70 3.54 ± 0.51 5 0.00 No 90 3.28 ± 0.36 5 0.49 No rSM94 0-10 4.44 ± 0.38 8 30 4.10 ± 0.45 8 0.58 No 50 3.84 ± 0.28 8 1.27 No 70 4.71 ± 0.48 8 0.44 No 90 4.04 ± 0.31 8 0.37 No a t was calculated from the formula (reference 9)
rSN103
0
0-10 30 50 70 90 0-10 30 50 70 90 0-10 30 50 70 90 0-10 30
000
C6 0'
06
FIG.
70
50
30
Time
without
replication
(min)
1. Results of holding experiments with six
rII
mutants. The figure shows the percentage of singly infected bacteria which produce mottled plaques after incubation for 10, 30, 50,
70, and 90
min
under
nonreplicating conditions. Values are plotted as the percentage of the mottled plaque frequency obtained without inhibition of DNA synthesis. Symbols: rEM84;
*,
*,
rSM94;
0, rSN1 03;
A\, rC204;
OJ,
*, rNT88;
rUV74.
TABLE 4. Burst size d uring FUdR-holding conditions"
t= (ic P (AX IUY)hyp EX2 +EY2 1 (n,_i) + (nu,-.) n,
1 nV wherei andy, are the average percentage of mottled plaques at 0 to 10 min and later times, respectively; £ and y2 are measures of the variance, and n is the number of trials. b The t values were evaluated at P = 0.05 with degrees of freedom = (2N - 2) (reference 10). c Percentage ± standard error.
from mixed infections, was determined as a function of time. The parental phage were prepared by growth individually in the presence of FUdR to make them like the phage in the heterozygote-enriched population. As shown in Table 5, there is no loss of segregating capacity, indicating that the FUdR holding conditions do not prevent detection of mixed yields. To examine the possible involvement of the
Titer of infective (min) (m) centers (10-6 phage/ml)
Avg burst size per producZero time tive infec- Zero time M
tivetercen-
(phage/ infective center)
89 2.0 0 55 49 75 1.5 10 24 27 60 1.2 30 33 30 60 1.2 50 33 30 65 1.3 70 28 25 65 1.3 90 a Cells were maintained under FUdR holding conditions for the indicated times and diluted to remove FUdR. Infective center assays were determined at the time of removal of FUdR. Burst size determinations were made after incubation for 90 min at 32°C in H-broth, followed by treatment with
CHCl3.
444
BERGER AND PARDOLL
J. VIROL.
repair process associated with the T4 v gene (4), we have examined segregation capacity using phage that also contain the v- gene mutation (6). The time-dependent loss of mottled plaques occurs with v- phage, indicating that v+ activity is not required for the recognition of singlebase mismatches (Fig. 2).
Figure 2 also shows the results of holding experiments carried out when phage-specific protein synthesis was prevented by addition of chloramphenicol 3 min postinfection. The loss of segregation capacity appears reduced under these conditions, indicating that repair of mismatched bases in T4 DNA may utilize both host and phage systems. TABLE 5. Lack of change in percentage of DISCUSSION segregating infective centers during "FUdR holding conditions" with mixedly infected cellsa The data presented indicate that there is a mottled Avgplaques' mechanism for recognition of cerpercentage site-specific Time (mm) (% of Time (min) t SD) tain mismatched bases in T4 DNA (Fig. 1). 3.8 ± 0.4 Correction of mismatched sites involves transi10 30 3.6 ± 0.2 tion and one possible transversion mutations 50 3.9 ± 0.2 (Table 1). Recognition of mismatches also may 3.8 ± 0.3 70 influenced by neighboring base sequences be 90 4.3 ± 0.2 since two mutant sites, rEM84 and rC204, are a1 The cells were infected with an average multi- tentatively classified as transition mutations plicity of infection of 0.1 of each parental phage,held with an adenine-thymine base pair at the muin FUdR at 42C for the indicated times, and plated tant site, yet only the rEM84 mutant shows on E. coli B. The frequency of mottled plaques was loss of heterozygosity. Loss of heterozygosity determined from coded plates after 12 to 16 h of still occurs in a v- genetic background, indicatincubation at 320C. ing that mismatch recognition is not mediated b Each average is based upon four separate determinations. The standard deviation of the mean is by endonuclease V, previously shown to be involved in the removal of pyrimidine dimers (5). indicated. Further studies are necessary to show that the loss of heterozygosity is part of a concerted process which results in a heterozygous to homozygous conversion. In the present study 110 , we cannot differentiate between recognition of mismatched sites as part of a process that re0 6 stores complementarity or to a process that results in preferential inactivation of heteroduplex DNA molecules. The S1 endonuclease of Aspergillus oryzae (1) acts at base mismatches CL in heteroduplex DNA produced in vitro (14), and in vivo repair of heteroduplex DNA re90. cently has been reported using transfection with X bacteriophage (19) and simian virus 40 *0 (C. J. Lai and D. Nathans, Fed. Proc. 34:515, 1975). As with T4, biochemical elucidation of the process in these cases awaits further experimentation. 6)
0
80
E
0
70-
0)
CP 60a.
Time without replication (min) FIG. 2. Holding experiments with populations enriched for rUV74 heteroduplex heterozygotes in a v+ genetic background = A, in a v- genetic background = 0, and in the presence of chloramphenicol (250 pgl ml) = A. The points which are statistically different from the 10 min time using the Student's t test (P = 0.05) are starred.
ACKNOWLEDGMENTS Nancy V. Hamlett, Richard P. Cunningham, and Philip E. Hartman assisted in the preparation of the final draft of this manuscript. This work was supported in part by Public Health Service research grants AI-08161 of the National Institute of Allergy and Infectious Diseases, GB-43825 of the National Science Foundation, and NP-175 of the American Cancer Society. H. Berger was a recipient of a Career Development Award, National Institutes of Health. This paper is contribution no. 863 of the Department of Biology, The Johns Hopkins University. LITERATURE CITED 1. Ando, T. 1966. A nuclease specific for heat-denatured DNA isolated from a product of Aspergillus oryzae. Biochim. Biophys. Acta 114:158-168.
VOL. 20, 1976 2. Champe, S. P., and S. Benzer. 1962. Reversal of mutant phenotypes by 5-fluorouracil: an approach to nucleotide sequence in messenger-RNA. Proc. Natl. Acad. Sci. U.S.A. 48:532-546. 3. Ephrussi-Taylor, H., and T. C. Gray. 1966. Genetic studies of recombining DNA in pneumococcal transformation. J. Gen. Physiol. 49:211-231. 4. Freese, E. B., and E. Freese. 1966. Induction of pure mutant clones by repair of inactivating DNA alterations in phage T4. Genetics 54:1055-1067. 5. Friedberg, E. C., and J. J. King. 1971. Dark repair of ultraviolet-irradiated deoxyribonucleic acid by bacteriophage T4: purification and characterization of a dimer specific phage-induced endonuclease. J. Bacteriol. 106:500-507. 6. Harm, W. 1963. Mutants of phage T4 with increased sensitivity to ultraviolet. Virology 19:.66-71. 7. Krisch, H. M., N. V. Hamlett, and H. Berger. 1972. Polynucleotide ligase in bacteriophage T4D recombination. Genetics 72:187-203. 8. Lacks, S. 1970. Mutants of Diplococcus pneumoniae that lack deoxyribonucleases and other activities possibly pertinent to genetic transformation. J. Bacteriol. 101:373-383. 9. Minium, E. W. 1970. Statistical reasoning in psychology and education. John Wiley and Sons, New York. 10. Mosig, G. 1970. Recombination in bacteriophage T4. Adv. Genet. 15:1-53. 11. Norkin, L. C. 1970. Marker specific effects in genetic
RECOGNITION OF MISMATCHED BASES
445
recombination. J. Mol. Biol. 51:633-655. 12. Roger, M. 1972. Evidence for conversion of heteroduplex transforming DNAs to homoduplexes by recipient pneumococcal cells. Proc. Natl. Acad. Sci. U.S.A. 69:466-470. 13. Sechaud, J., G. Streisinger, J. Emrich, J. Newton, H. Lanford, H. Reinhold, and M. M. Stahl. 1965. Chromosome structure in phage T4. II. Terminal redundancy and heterozygosis. Proc. Natl. Acad. Sci. U.S.A. 54:1333-1339. 14. Shenk, T. E., C. Rhodes, P. W. J. Rigby, and P. Berg. 1975. Biochemical method for mapping mutational alterations in DNA with S1 nuclease: the location of deletions and temperature-sensitive mutations in Simian virus 40. Proc. Natl. Acad. Sci. U.S.A. 72:989-993. 15. Spatz, H. C., and T. A. Trautner. 1970. One way to do experiments on gene conversion? Transfection with heteroduplex SPP1 DNA. Mol. Gen. Genet. 109:84106. 16. Stadler, D. 1973. The mechanism of intragenic recombination. Annu. Rev. Genet. 7:113-127. 17. Summers, G. A., and J. W. Drake. 1971. Bisulfite mutagenesis in bacteriophage T4. Genetics 68:603-07. 18. Tessman, I. 1965. Genetic ultrafine structure in the T4 rII region. Genetics 51:63-75. 19. Wildenberg, J., and M. Meselson. 1975. Mismatch repair in heteroduplex DNA. Proc. Natl. Acad. Sci. U.S.A. 72:2202-2206.
Vol. 20, No. 2 Printed in U.S.A.
Copyright C 1976 American Society for Microbiology
Evidence That Mismatched Bases in Heteroduplex T4 Bacteriophage Are Recognized In Vivo' HILLARD BERGER2 AND DREW PARDOLL3 Department ofBiology, The Johns Hopkins University, Baltimore, Maryland 21218 Received for publication 1 June 1976
T4 heteroduplex heterozygotes are lost selectively after prolonged incubation of phage-infected Escherichia coli cells under nonreplicating conditions. The loss of heterozygosity occurs for four out of six ril sites tested and is not dependent upon T4 v gene function. The results are interpreted to indicate the existence of a base-specific system for the recognition of mismatched bases in intracellular DNA.
Many models for genetic recombination postulate the existence of a mechanism for the repair of mismatched bases in heteroduplex DNA. Genetic phenomena which may be explained by mechanisms for "prereplicative restoration" of complementarity in heteroduplex DNA are: (i) gene conversion in fission fungi and Ascobulus immersus (see reference 16 for a review), (ii) high negative interference observed in multifactor phage crosses (reviewed in reference 10), (iii) short distance marker effects in phage and bacterial crosses (11, 18), and (iv) low integration efficiency in transformation (3, 8). Genetic and physicochemical experiments have established that heteroduplex DNA is an intermediate in recombination (reviewed in reference 10). Recent experiments with Bacillus subtilis phage SPP 1 (15), Escherichia coli phage X (19), and Pneumococcus (12) indicate that the DNA in these systems is subject to mismatch correction; however, data indicating that heteroduplex DNA is subject to site-specific repair is lacking in the bacteriophage T4 system. To investigate this point we have singly infected cells with T4 phage populations enriched for heteroduplex heterozygotes (13) and examined these infected cells for loss of segregating capacity (mottled plaques) under nonreplicating conditions. The results indicate: (i) after prolonged incubation under nonreplicating conditions, the capacity of heterozygous T4 phage to segregate is reduced, (ii) this apparent recognition of heterozygous DNA is marker-specific, (iii) the apparent repair is not dependent on the ' Address reprint requests to: Chairman, Department of Biology, The Johns Hopkins University, Baltimore, MD 21218. 2 Deceased 28 October 1975. 3 Present address: The Johns Hopkins School of Medicine, Baltimore, MD 21205.
v gene-encoded endonuclease V (5), and (iv) the process is partially dependent upon phage-specific protein synthesis. MATERIALS AND METHODS Bacteria. E. coli B was used as the host for all crosses and assays, except for determination of reversions to rIl+, in which case K-12(X) was used as indicator. Phage mutants. The rH mutants used have been previously described and are derived from T4B (2, 17). T4D tsG40 (gene 431) was repeatedly backcrossed to T4B and used to make double mutants. The v gene mutant was originally obtained from W. Harm (6) and was extensively backcrossed to T4B before use in the construction of the v-rIl-ts and v-ts mutants. Preparation of phage stocks enriched in heterozygotes. Phage stocks containing 4 to 5% heterozygotes for each of six different rHI sites were prepared by crossing tsG40-rII phage with tsG40-rII+ phage under conditions in which DNA synthesis was inhibited by addition of 5-fluorodeoxyuridine (FUdR) (13). Crosses were done at 30'C, and the plates were incubated from 12 to 16 h at 32'C before scoring for mottled plaques. All scoring was from coded plates to prevent bias. Holding experiments. Phage stocks enriched for heterozygotes were used to infect E. coli B at 3 x 108 cells/ml, with a multiplicity of infection of 0.01. Infection was done in M9+ medium supplemented with FUdR (20 ug/ml), uracil (40 Ag/ml), and Ltryptophan (20 ,ug/ml) at 42°C. Under these conditions there was no measurable DNA replication (H. Berger, unpublished data). At 5 min after infection the culture was diluted 50-fold into the same medium containing anti-T4 serum. The antiserum was added to inactivate unadsorbed phage and to inactivate any rare phage released during the holding period. The antiserum coupled with the dilution from the initial adsorption mixture insured that only primary infective centers would be scored upon plating after the hold. At various times the hold was terminated by dilution and plating at 32°C. The frequency of infected cells producing mottled 441
442
J. VIROL.
BERGER AND PARDOLL
plaques was determined after 12 to 15 h of incubation from coded plates. When chloramphenicol was used it was added 3 min after infection to a final concentration of 250 ,ug/ml. Approximately 50 mottled plaques were scored for each determination. The average percentage of mottled plaques was based on at least five and as many as eight individual trials. Media. The media, preparation of indicator bacteria, and standard cross procedures to prepare multiple mutants are as described by Krisch et al. (7).
RESULTS The rlI mutants used in this study have been characterized by their response to specific mutagens (2, 17). A summary of tentative base pair assignments and type of mutation is shown in Table 1. Also included are the results of the reversion response to 5-bromodeoxyuridine performed in conjunction with the present study. These results confirm the previous findings, with the exception of rSM94, which was previously classified as a transversion mutation but which appears to be a transition change. As described in Materials and Methods, each of the rII-tsG40 double mutants was crossed with tsG40 in the presence of FUdR to produce a phage population enriched for heterozygotes. Table 2 shows the average percentage of heterozygotes (mottled plaques) and yield obtained from these crosses. Since all of the phage contained the tsG40 mutation, plates were incubated at 32°C before mottled plaques were scored. The heterozygote-enriched populations
TABLE 2. Preparation of phage stocks enriched for heterozygotes Cross Cross
rSN103-tsG40 x tsG40 rC204-tsG40 x tsG40 rNT88-tsG40 x tsG40 rUV74-tsG40 x tsG40 rEM84-tsG40 x tsG40 rSM94-tsG40 x tsG40
Burst size Mottled(%) fected (phage/inbacteplaques rium)
4.8 5.0 4.3 5.4 4.0 4.3
1.1 1.8 0.7 0.8 2.1 2.0
were then used to infect E. coli B in the presence of FUdR at the nonpermissive temperature. The infected cells were held under these nonreplicating conditions for various times, plated under permissive conditions, and scored for the frequency of infected cells capable of rIl segregation as measured by the appearance of mottled plaques. The data are shown in Table 3 and graphically depicted in Fig. 1. For four of the six sites (mutants rSN103, rC204, rNT88, and rUV74) there is a time-dependent decrease in frequency of infective centers containing segregants, significant at P = 0.05 for late sampling times (9). Possible explanations for the time-dependent loss in segregating capacity are: (i) the operation of a site-specific repair process; (ii) a generalized inactivation of DNA strands; (iii) random repair similar to the process described by Freese and Freese (4) to explain UV-stimulated production of pure clones after hydroxylamine treatment of T4 phage; (iv) incomplete genomic TABLE 1. Base substitutions in rII mutants sampling attributable to decreased burst sizes resulting from the maintenance of infected cells Response under nonreplicating conditions for extended to Base Misperiods. Each of the latter three explanations BUdR match Mutant Most likely pair at Referpredicts a decline in heterozygosity for all rIl mutant type mutant ence (muta- repairc markers. In contrast, as shown in Table 3 and gen/ site a spontain Fig. 1, the frequency of segregating infective neous)" centers remains nearly constant for the rSM94 + 56 2 and rEM84 mutations. We have also measured rSN103 Transition G*C 7 2 rEM84 Transition A-T the effect of the FUdR holding conditions on + 2 2 rC204 Transition A-T average burst size (Table 4). Although pro+ 56 2 rNT88 Transition G * C longed maintenance of infected cells under non+ 3 18 rUV74d Transver- A-T replicating conditions does decrease phage sion? yields, the average phage production per pro65 18 rSM94d Transition G C ductive infective center is sufficiently large to a Abbreviations: G- C, Guanine -cytosine, A-T, suggest that all genomic types would be inadenine-thymine. cluded in an individual yield. To further show b Revertants were measured by plating on B and that mixed yields are efficiently recognized unK12 (X) after one cycle of growth in the presence of der the FUdR holding conditions, we have car100 ,ug of BUdR per ml in M9+ medium. set of reconstruction experiments. Data of this paper; +, present; -, none detected. ried out aFUdR holding conditions, cells were Using the d These mutations previously were classified as transversions. Mutation rSM94 responds as a tran- infected with an average multiplicity of infecsition mutation, whereas rUV74 gives a questiona- tion of 0.1 of tsG40 and tsG40-rUV74 phage and the frequency of mottled plaques, which arise ble response in our tests. c
443
RECOGNITION OF MISMATCHED BASES
VOL. 20, 1976 TABiE 3. Effects ofprolonged incubation without DNA synthesis on the segregation capacity of heteroduplex heterozygotes for six rI point-mutation sites Significantly
Hold-
Avg percent- N f ing age of mottled Marker time plaques (% ± trials (n) SE) (min)
ta
different from "010" trialb (P
=
0.05)
5.04 + 0.36 5 4.62 ± 0.11 5 1.05 No 3.84 ± 0.22 5 2.77 Yes 1.77 No 4.10 ± 0.39 5 3.80 ± 0.27 5 3.81 Yes 4.96 ± 0.25 5 rC204 4.34 ± 0.08 5 2.34 Yes 3.60 ± 0.08 5 5.17 Yes 3.58 + 0.24 5 3.50 Yes 3.40 ± 0.23 5 4.56 Yes 4.03 ± 0.40 8 rNT88 3.45 ± 0.29 8 1.17 No 3.28 + 0.22 8 1.64 No 2.80 + 0.21 8 2.70 Yes 2 54 0.26 8 3.10 Yes 5.30 ± 0.30 7 rUV74 4.90 + 0.37 7 0.83 No 50 4.39 + 0.39 7 1.84 Yes 70 3.94 ± 0.29 7 3.16 Yes 90 3.78 ± 0.24 7 4.83 Yes rEM84 0-10 3.54 ± 0.40 5 30 3.70 ± 0.40 5 0.48 No 50 3.28 ± 0.37 5 0.48 No 70 3.54 ± 0.51 5 0.00 No 90 3.28 ± 0.36 5 0.49 No rSM94 0-10 4.44 ± 0.38 8 30 4.10 ± 0.45 8 0.58 No 50 3.84 ± 0.28 8 1.27 No 70 4.71 ± 0.48 8 0.44 No 90 4.04 ± 0.31 8 0.37 No a t was calculated from the formula (reference 9)
rSN103
0
0-10 30 50 70 90 0-10 30 50 70 90 0-10 30 50 70 90 0-10 30
000
C6 0'
06
FIG.
70
50
30
Time
without
replication
(min)
1. Results of holding experiments with six
rII
mutants. The figure shows the percentage of singly infected bacteria which produce mottled plaques after incubation for 10, 30, 50,
70, and 90
min
under
nonreplicating conditions. Values are plotted as the percentage of the mottled plaque frequency obtained without inhibition of DNA synthesis. Symbols: rEM84;
*,
*,
rSM94;
0, rSN1 03;
A\, rC204;
OJ,
*, rNT88;
rUV74.
TABLE 4. Burst size d uring FUdR-holding conditions"
t= (ic P (AX IUY)hyp EX2 +EY2 1 (n,_i) + (nu,-.) n,
1 nV wherei andy, are the average percentage of mottled plaques at 0 to 10 min and later times, respectively; £ and y2 are measures of the variance, and n is the number of trials. b The t values were evaluated at P = 0.05 with degrees of freedom = (2N - 2) (reference 10). c Percentage ± standard error.
from mixed infections, was determined as a function of time. The parental phage were prepared by growth individually in the presence of FUdR to make them like the phage in the heterozygote-enriched population. As shown in Table 5, there is no loss of segregating capacity, indicating that the FUdR holding conditions do not prevent detection of mixed yields. To examine the possible involvement of the
Titer of infective (min) (m) centers (10-6 phage/ml)
Avg burst size per producZero time tive infec- Zero time M
tivetercen-
(phage/ infective center)
89 2.0 0 55 49 75 1.5 10 24 27 60 1.2 30 33 30 60 1.2 50 33 30 65 1.3 70 28 25 65 1.3 90 a Cells were maintained under FUdR holding conditions for the indicated times and diluted to remove FUdR. Infective center assays were determined at the time of removal of FUdR. Burst size determinations were made after incubation for 90 min at 32°C in H-broth, followed by treatment with
CHCl3.
444
BERGER AND PARDOLL
J. VIROL.
repair process associated with the T4 v gene (4), we have examined segregation capacity using phage that also contain the v- gene mutation (6). The time-dependent loss of mottled plaques occurs with v- phage, indicating that v+ activity is not required for the recognition of singlebase mismatches (Fig. 2).
Figure 2 also shows the results of holding experiments carried out when phage-specific protein synthesis was prevented by addition of chloramphenicol 3 min postinfection. The loss of segregation capacity appears reduced under these conditions, indicating that repair of mismatched bases in T4 DNA may utilize both host and phage systems. TABLE 5. Lack of change in percentage of DISCUSSION segregating infective centers during "FUdR holding conditions" with mixedly infected cellsa The data presented indicate that there is a mottled Avgplaques' mechanism for recognition of cerpercentage site-specific Time (mm) (% of Time (min) t SD) tain mismatched bases in T4 DNA (Fig. 1). 3.8 ± 0.4 Correction of mismatched sites involves transi10 30 3.6 ± 0.2 tion and one possible transversion mutations 50 3.9 ± 0.2 (Table 1). Recognition of mismatches also may 3.8 ± 0.3 70 influenced by neighboring base sequences be 90 4.3 ± 0.2 since two mutant sites, rEM84 and rC204, are a1 The cells were infected with an average multi- tentatively classified as transition mutations plicity of infection of 0.1 of each parental phage,held with an adenine-thymine base pair at the muin FUdR at 42C for the indicated times, and plated tant site, yet only the rEM84 mutant shows on E. coli B. The frequency of mottled plaques was loss of heterozygosity. Loss of heterozygosity determined from coded plates after 12 to 16 h of still occurs in a v- genetic background, indicatincubation at 320C. ing that mismatch recognition is not mediated b Each average is based upon four separate determinations. The standard deviation of the mean is by endonuclease V, previously shown to be involved in the removal of pyrimidine dimers (5). indicated. Further studies are necessary to show that the loss of heterozygosity is part of a concerted process which results in a heterozygous to homozygous conversion. In the present study 110 , we cannot differentiate between recognition of mismatched sites as part of a process that re0 6 stores complementarity or to a process that results in preferential inactivation of heteroduplex DNA molecules. The S1 endonuclease of Aspergillus oryzae (1) acts at base mismatches CL in heteroduplex DNA produced in vitro (14), and in vivo repair of heteroduplex DNA re90. cently has been reported using transfection with X bacteriophage (19) and simian virus 40 *0 (C. J. Lai and D. Nathans, Fed. Proc. 34:515, 1975). As with T4, biochemical elucidation of the process in these cases awaits further experimentation. 6)
0
80
E
0
70-
0)
CP 60a.
Time without replication (min) FIG. 2. Holding experiments with populations enriched for rUV74 heteroduplex heterozygotes in a v+ genetic background = A, in a v- genetic background = 0, and in the presence of chloramphenicol (250 pgl ml) = A. The points which are statistically different from the 10 min time using the Student's t test (P = 0.05) are starred.
ACKNOWLEDGMENTS Nancy V. Hamlett, Richard P. Cunningham, and Philip E. Hartman assisted in the preparation of the final draft of this manuscript. This work was supported in part by Public Health Service research grants AI-08161 of the National Institute of Allergy and Infectious Diseases, GB-43825 of the National Science Foundation, and NP-175 of the American Cancer Society. H. Berger was a recipient of a Career Development Award, National Institutes of Health. This paper is contribution no. 863 of the Department of Biology, The Johns Hopkins University. LITERATURE CITED 1. Ando, T. 1966. A nuclease specific for heat-denatured DNA isolated from a product of Aspergillus oryzae. Biochim. Biophys. Acta 114:158-168.
VOL. 20, 1976 2. Champe, S. P., and S. Benzer. 1962. Reversal of mutant phenotypes by 5-fluorouracil: an approach to nucleotide sequence in messenger-RNA. Proc. Natl. Acad. Sci. U.S.A. 48:532-546. 3. Ephrussi-Taylor, H., and T. C. Gray. 1966. Genetic studies of recombining DNA in pneumococcal transformation. J. Gen. Physiol. 49:211-231. 4. Freese, E. B., and E. Freese. 1966. Induction of pure mutant clones by repair of inactivating DNA alterations in phage T4. Genetics 54:1055-1067. 5. Friedberg, E. C., and J. J. King. 1971. Dark repair of ultraviolet-irradiated deoxyribonucleic acid by bacteriophage T4: purification and characterization of a dimer specific phage-induced endonuclease. J. Bacteriol. 106:500-507. 6. Harm, W. 1963. Mutants of phage T4 with increased sensitivity to ultraviolet. Virology 19:.66-71. 7. Krisch, H. M., N. V. Hamlett, and H. Berger. 1972. Polynucleotide ligase in bacteriophage T4D recombination. Genetics 72:187-203. 8. Lacks, S. 1970. Mutants of Diplococcus pneumoniae that lack deoxyribonucleases and other activities possibly pertinent to genetic transformation. J. Bacteriol. 101:373-383. 9. Minium, E. W. 1970. Statistical reasoning in psychology and education. John Wiley and Sons, New York. 10. Mosig, G. 1970. Recombination in bacteriophage T4. Adv. Genet. 15:1-53. 11. Norkin, L. C. 1970. Marker specific effects in genetic
RECOGNITION OF MISMATCHED BASES
445
recombination. J. Mol. Biol. 51:633-655. 12. Roger, M. 1972. Evidence for conversion of heteroduplex transforming DNAs to homoduplexes by recipient pneumococcal cells. Proc. Natl. Acad. Sci. U.S.A. 69:466-470. 13. Sechaud, J., G. Streisinger, J. Emrich, J. Newton, H. Lanford, H. Reinhold, and M. M. Stahl. 1965. Chromosome structure in phage T4. II. Terminal redundancy and heterozygosis. Proc. Natl. Acad. Sci. U.S.A. 54:1333-1339. 14. Shenk, T. E., C. Rhodes, P. W. J. Rigby, and P. Berg. 1975. Biochemical method for mapping mutational alterations in DNA with S1 nuclease: the location of deletions and temperature-sensitive mutations in Simian virus 40. Proc. Natl. Acad. Sci. U.S.A. 72:989-993. 15. Spatz, H. C., and T. A. Trautner. 1970. One way to do experiments on gene conversion? Transfection with heteroduplex SPP1 DNA. Mol. Gen. Genet. 109:84106. 16. Stadler, D. 1973. The mechanism of intragenic recombination. Annu. Rev. Genet. 7:113-127. 17. Summers, G. A., and J. W. Drake. 1971. Bisulfite mutagenesis in bacteriophage T4. Genetics 68:603-07. 18. Tessman, I. 1965. Genetic ultrafine structure in the T4 rII region. Genetics 51:63-75. 19. Wildenberg, J., and M. Meselson. 1975. Mismatch repair in heteroduplex DNA. Proc. Natl. Acad. Sci. U.S.A. 72:2202-2206.
