VIROLOGY
65, 281-285 (1975)
The Involvement
of Genes 3, 4, 5 and 6 in Genetic
Recombination CAROL Departments
of Pathology
KERR
and Medical
in Bacteriophage AND
PAUL
D. SADOWSKP
Genetics University
Accepted January
T7 l
of Toronto,
Toronto,
M5S lA8, Canada
8, 1975
The effect of conditionally lethal mutations in various genes of phage T7 on genetic recombination was determined by doing genetic crosses under nonpermissive conditions. The experiments have implicated the products of gene 3 (T7 endonuclease), gene 4 (DNA replication protein), gene 5 (T7 DNA polymerase) and gene 6 (T7 exonuclease) in phage T7 genetic recombination.
During recent years a great deal of information has accumulated concerning DNA metabolism after phage T7 infection (I). Degradation of the host chromosome requires action of the gene 3 product, T7 endonuclease I (2-5), and the gene 6 product, T7 exonuclease (6-8). Early after infection, phage DNA replication begins at a site about 17% from the genetic left end and proceeds bidirectionally (9-1 I), whereas late in the infectious cycle replicating DNA can be isolated as structures longer than mature phage DNA (12-15). T7 DNA replication requires the action of the gene 5 product, a T7-induced DNA polymerase (16, 17), the gene 4 product required for synthesis of DNA strands that are complementary to L strands (18), and the gene 2 product whose function is unknown (I). The phage also induces the synthesis of a phage-coded, ATP-requiring DNA ligase (19) whose function in DNA replication can be assumed by the host ligase. Some of the above products have been shown to have dual functions. Thus both T7 endonuclease I and T7 exonuclease are required for maturation of phage DNA in addition to their role in the destruction of ‘Supported by the Medical Research Council of Canada. 2Medical Research Council of Canada Associate; to whom correspondence should be addressed.
host DNA (15). Because of the possibility that some phage products might subserve more than one function, we have studied the effects of temperature-sensitive missense and amber nonsense mutations in genes 2-6 upon genetic recombination. We have found that the products of genes 3, 4, 5 and 6 are involved in genetic recombination. All T7 amber mutants were from the collection of F. W. Studier (20) and most have been listed previously (21). Additional amber mutants from Studier were am 290 (gene 17), am lys 3-l (gene 3.5), am 20 (gene 4), am 233 and am 111 (both in gene 6). Temperature-sensitive mutants were also obtained from F. W. Studier and included ts 106 and ts 136 (both in gene 6), ts 11 (gene 5), ts 101 (gene 4), and ts 105 (gene 19). Escherichia coli 011’ and E. coli B were the permissive and nonpermissive strains, respectively, for amber mutants (20). The following recombination-deficient hosts were used: E. coli QR 48 (recA), E. coli AB 2470 (recBBl), E. coli JC 5495 (recA13, recB21) and E. coli JC 5547 (recA13, recB21, recC22) (22). These strains were obtained from A. Becker and D. Hoar of this department. Suitably marked strains were constructed by standard phage crosses (20) and tested by complementation (20). The crosses that tested recombination profi281
Copyright All rights
0 1975 hy Academic Press, of reproduction in any form
Inc. reserved.
282
SHORT
COMMUNICATIONS
ciency were done essentially according to tage of enabling us to determine that a Studier’s method (20) with the following recombinational event yielded the two rein approximately equal modifications. The cells were grown in combinants tryptone broth which had been suppleamounts. mented with 1 mM MgSO, to aid phage We were interested in determining which adsorption. The phage and cells were T7-coded functions were involved in phage shaken manually in an adsorption tube for genetic recombination. Prior to doing this, 5 min and then centrifuged at 10,000 g for 5 it was important to establish that T7 min to remove unadsorbed phage. The titer genetic recombination did not depend on and genotype of the unadsorbed phage the major host recombination system. present in the supernatant fluid were de- From the results in Table 1, it is apparent termined. The pellet was resuspended in that T7 recombination between amber 0.8 ml of broth containing 4 mM KCN, and markers in genes 9 and 15 is not inhibited an aliquot was plated for infectious cen- by mutations in the recA, -B or -C genes. If phage recombination is someters. The remainder was diluted 5 x 105- anything, fold into broth and the final dilution was what increased in the absence of ret function. incubated at the appropriate temperature for 1 hr. The total burst was determined by We then proceeded to examine the efplating on the permissive host, E. coli 011’. fects of amber mutations in various genes upon recombination frequencies between In all crosses, the percentage of unadsorbed amber markers in genes 9 and 15. Reprewas less than 15%. The reversion frequency of single amber mutations used was less sentative results of several experiments are in Table 2. The results are than 10-4, a figure too low to influence the summarized percentage of wild-type recombinants ob- expressed as a ratio of the recombination frequency obtained for the amber mutation tained. in question over the frequency for the Recombination frequencies were determined as follows. When we wished to test amber+ allele (am/am+, final column). A decrease in this ratio in the nonpermissive the effect of a temperature-sensitive mutation on recombination between two amber host (B) would tend to implicate the mutated gene in recombination. The results markers (an, and am,), we constructed phages bearing the amber mutation and may be summarized as follows. Gene 10 the temperature-sensitive mutation in (head protein), gene 17 (tail protein), gene question (i.e., am,, ts and am,, ts). Crosses 3.5 (lysozyme) and gene 2 (reduced DNA were carried out between am, and am2 and synthesis) do not appear to be involved in genetic recombination (Expt 1-4, Table 2). between am, ts and am2 ts at temperatures ranging from 30 to 42”. The percent recomTABLE 1 binants in each case could be readily deterRECOMBINATION OF PHAGE T7 IN Ret’ AND Ret HOSTS” mined by plating for am+ recombinants on E. coli B at the permissive temperature. 5% Host Genotype Burst size Rf?COIiThis figure was doubled to account for bination reciprocal recombinants. If we wished to determine the effect of an 19.2 011’ sup- ret+ 134 amber mutation, am3, upon recombination sup ret+ 118 21.5 25.6 :R48 recA, sup 71 between am, and am, markers, we carried AB2470 recB, sup30.4 69 out a cross of the type am, am3 x am, am3 30.8 JC5495 recAB, sup+ 71 in both the permissive host (011’) and the recABC, sup+ 71 26.3 JC5547 nonpermissive host (B). In this cross, both parental phages contained two amber mua Recombination was measured between amber tations so that it was necessary to determarkers in genes 9 and 15 by measuring the percentmine the genotypes of the progeny by age of am+ recombinants among the progeny and complementation tests. This procedure, doubling this percentage to account for the reciprocal recombinants. although laborious, had the added advan-
SHORT
283
COMMUNICATIONS TABLE
2
EFFECTS OF VARIOUS AMBER MUTATIONS ON GENETIC RECOMBINATION IN PHAGE T7” Expt
Amber
Gene tested
Function affected
Host
am Burst per cell
1
13
10
Head protein
2
290
17
Tail protein
3
lys 3-1
3.5
Lysozyme
4
64
2
5
29
3
Defective DNA synthesis T7 endonuclease
6
20
4
DNA negative
7
28
5
T7 DNA polymerase
8
233
6
T7 exonuclease
I
011’ B 011’ B 011’ B 011’ B 011’ B 011’ B 011’ B 011’ B
168 2.7 131 4.8 295 11.7 305 3 280 1.7 188 1.5 298 0.93 202 1
am+
7% Recombination 24.5 15.6 27.9 21.6 13.8 14 36.3 26.2 30.6 7.2 32.2 2.8 29.6 3.5 27.5 3.8
Burst per cell 252 124 250 136 340 240 176 218 225 101 370 207 296 179 122 115
% Recombination 20 14.1 29.2 26.6 27.2 25.4 26.6 23.2 20 22.6 30.8 25.6 21.8 17.6 26.4 24.2
amlam + Relative recombination 1.2 1.1 0.96 0.81 0.51 0.55 1 1.1 1.5 0.3 1.05 0.11 1.36 0.2 1.04 0.16
” Recombination was determined between amber markers in genes 9 and 15. Results are given for the amber mutation tested (“am”) and the controls which lacked that amber mutation (“am+“).
These data show that normal recombination frequencies are obtainable even though the burst size is significantly reduced. On the other hand, mutations in genes 3-6 resulted in greatly reduced recombination when the crosses were done in a suphost (Expt 5-8, Table 2). These effects were gene specific since mutations at different sites in the same gene gave identical results. In addition, the use of urn+ revertants of the mutants tested caused recombination rates to revert to normal (data not shown). Finally, the reduced recombination effects were not dependent upon the markers between which recombination was measured in that identical results were obtained when recombination was measured between markers in genes 15 and 17 or genes 8 and 18. Having obtained suggestive evidence for the involvement of certain genes in recombination from experiments using amber mutations, we attempted to confirm these results using temperature-sensitive (ts) mutations. The ts mutation to be tested was therefore crossed into the 9am and
15am phage and the recombination frequency between these two amber markers was measured as a function of temperature. In a control experiment, a ts mutation in gene 19 (a function involved in phage DNA maturation) caused at most a limited decrease in recombination at high temperatures, while the burst size relative to the ts+ phage was reduced almost lOOO-fold (Fig. la). Temperature-sensitive mutations in gene 4 (Fig. lb), gene 5 (Fig. lc) and gene 6 (Fig. Id) caused much more marked decreases in recombination than the ts mutation in gene 19. One could argue that the effects seen using both am and ts mutations are simply due to the low burst sizes which result when the crosses are performed under nonpermissive conditions. To answer this objection, we measured genetic recombination in the presence of nalidixic acid, a drug that inhibits both phage production and DNA synthesis. As can be seen in Fig. 2, there is relatively little effect of the drug on genetic recombination, even at doses of nalidixic acid that reduce the burst sizes to the levels seen in our experiments (Table 2,
284
SHORT COMMUNICATIONS
01
001 26
30
34
38
42
Temperature
26
30
34
38
42
(‘C)
FIG. 1. Effects of ts mutations in various genes upon genetic recombination. E. coli 011’ was infected with phage bearing the ts mutations to be tested as well as amber mutations in gene 9 or 15 (the markers between which recombination is measured) at temperatures ranging from 26-42”. Recombinants were scored as am+ phage plated at room temperature. Genes tested: (a) gene 19; (b) gene 4; (c) gene 5; (d) gene 6. *-----*, Relative burst (ts/ts+); O---O, % recombination, control (ts’); O-O, % recombination, test (ts).
Fig. 1). A level of 30 pg per ml of nalidixic acid reduces the rate of T7 DNA synthesis by greater than 99% (data not shown). Thus it is unlikely that the effects of mutations in genes 3-6 are due to the nonspecific decrease in burst size. A second possibility which we cannot exclude is that DNA replication is required for recombination. The products of genes
3-6 are required for normal rates of DNA synthesis (20). If DNA replication and recombination are inseparable, then a deficiency of any of these gene products would decrease recombination rates. Although nalidixic acid abolished more than 99% of DNA synthesis without affecting recombination rates appreciably, a limited amount of DNA replication, sufficient to allow
SHORT
285
COMMUNICATIONS REFERENCES
1. STUDIER, F. W., Science 176, 367-376 (1972). 2. CENTER, M. S., STUDIER, F. W., and RICHARDSON,
C. C., hoc. Nat. Acad. Sci. USA 66, 242-248
Frc. 2. Effect of nalidixic acid on burst size and genetic recombination in phage T7. E. coli B was infected with ten phage per cell each of T7 am 9 and T7 am 15 in the presence of increasing concentrations of nalidixic acid at 30”. After 60 min. the percentage of recombinants was determined by measuring the proportion of,am+ progeny over total progeny. *-----*, burst per cell; O-0, ‘% recombination.
recombination may have occurred. Indeed, in phage X, Stahl and Stahl (23, 24) have found that recombination in most regions of the chromosome is accompanied by considerable DNA replication. These experiments suggest that some T7 gene products play a multifunctional role in DNA metabolism. T7 endonuclease I (gene 3 product) and the T7 gene 6 exonuclease have already been implicated in destruction of cellular DNA and phage DNA maturation. A recent paper has also implicated the exonuclease in genetic recombination (25). It is relatively easy to conceive of a role for an endonuclease and exonuclease in a breakage and reunion mechanism of genetic recombination (26). In addition to its role in DNA replication, the T7 DNA polymerase may contribute to the repair of recombinant molecules or its 3’ exonuclease may also be involved in trimming back redundant single strands. The role of the gene 4 product in recombination is unknown. These experiments show that at least four T7 gene products are involved in genetic recombination. The availability of all of these products in purified form (3, 5, 6, 16-18) should facilitate attempts to perform genetic recombination with T7 DNA in vitro.
(1970). 3. CENTER, M. S., and RICHARDSON, C. C., J. Biol. Chem. 245, 6285-6291 (1970). 4. CENTER, M. S., and RICHARDSON, C. C., J. Biol. Chem. 245, 6292-6299 (1970). 5. SADOWSKI, P. D., J. Biol. Chem. 246, 209-216 (1971). 6. KERR, C., and SADOWSKI, P. D., J. Biol. Chem. 247, 305-310 (1972). 7. KERR, C., and SADOWSKI, P. D., J. Biol. Chem. 247, 311-318 (1972). 8. SADOWSKI, P. D., and KERR, C., J. Virol. 6, 149-155 (1970). 9. WOLFSON, J., DRESSLER, D., and MAGAZIN, M., Proc. Nat. Acad. Sci. USA 69, 499-504 (1972). IO. DRESSLER, D., WOLFSON, J., and MAGAZIN, M., Proc. Nat. Acad. Sci. USA 69,9981002 (1972). 11. WOLFSON, J., and DRESSLER,D., Proc. Nat. Acad. Sci. USA 69,2682-2686 (1972). 12. KELLY, T. J., and THOMAS, C. A., JR., J. Mol. Biol. 44, 459-475 (1969). 13. SCHLEGEL,R. A., and THOMAS, C. A., JR., J. Mol. Biol. 68, 319-345 (1972). 14. WATSON, J. D., Nature New Biol. 239, 1977201 (1972). 15. STR~TLING, W., KRAUSE, E., and KNIPPERS, R., Virology 51, 109-119 (1973). 16. GRIPPO, P., and RICHARDSON,C. C., J. Biol. Chem. 246, 6867-6873 (1971). 17. OEY, J. L., STRXTLING, W., and KNIPPERS, R., Eur. J. Biochem. 23, 497-504 (1971). 18. STRXTLING, W., and KNIPPERS, R., Nature (London) 245, 195-197 (1973). 19. MASAMUNE, Y., FRENKEL, G. D., and RICHARDSON, C. C., J. Biol. Chem. 246,6874-6879 (1971). 20. STUDIER, F. W., Virology 39, 562-574 (1969). 21. SADOWSKI,P. D., Can. J. Biochem. 50, 1016-1023 (1972). 22. BACHMANN, B. J., Bacterial. Reo. 36, 525-557 (1972). 23. STAHL, M. M., and STAHL, F. W., In “The Bacteriophage Lambda” (A. D. Hershey, Ed.) p. 431-442 (Cold Spring Harbor Laboratory) (1971). 24. STAHL, F. W., and STAHL, M. M., In “The Bacteriophage Lambda” (A. D. Hershey, Ed.) pp. 443-453. Cold Spring Harbor Laboratory Cold Spring Harbor, NY, 1971. 25. LEE, M., and MILLER, R. C., JR., J. Virol. 14, 1040-1048 (1974). 26. BROKER, T. R., and LEHMAN, I. R., J. Mol. Biol. 60, 131-149 (1971).
65, 281-285 (1975)
The Involvement
of Genes 3, 4, 5 and 6 in Genetic
Recombination CAROL Departments
of Pathology
KERR
and Medical
in Bacteriophage AND
PAUL
D. SADOWSKP
Genetics University
Accepted January
T7 l
of Toronto,
Toronto,
M5S lA8, Canada
8, 1975
The effect of conditionally lethal mutations in various genes of phage T7 on genetic recombination was determined by doing genetic crosses under nonpermissive conditions. The experiments have implicated the products of gene 3 (T7 endonuclease), gene 4 (DNA replication protein), gene 5 (T7 DNA polymerase) and gene 6 (T7 exonuclease) in phage T7 genetic recombination.
During recent years a great deal of information has accumulated concerning DNA metabolism after phage T7 infection (I). Degradation of the host chromosome requires action of the gene 3 product, T7 endonuclease I (2-5), and the gene 6 product, T7 exonuclease (6-8). Early after infection, phage DNA replication begins at a site about 17% from the genetic left end and proceeds bidirectionally (9-1 I), whereas late in the infectious cycle replicating DNA can be isolated as structures longer than mature phage DNA (12-15). T7 DNA replication requires the action of the gene 5 product, a T7-induced DNA polymerase (16, 17), the gene 4 product required for synthesis of DNA strands that are complementary to L strands (18), and the gene 2 product whose function is unknown (I). The phage also induces the synthesis of a phage-coded, ATP-requiring DNA ligase (19) whose function in DNA replication can be assumed by the host ligase. Some of the above products have been shown to have dual functions. Thus both T7 endonuclease I and T7 exonuclease are required for maturation of phage DNA in addition to their role in the destruction of ‘Supported by the Medical Research Council of Canada. 2Medical Research Council of Canada Associate; to whom correspondence should be addressed.
host DNA (15). Because of the possibility that some phage products might subserve more than one function, we have studied the effects of temperature-sensitive missense and amber nonsense mutations in genes 2-6 upon genetic recombination. We have found that the products of genes 3, 4, 5 and 6 are involved in genetic recombination. All T7 amber mutants were from the collection of F. W. Studier (20) and most have been listed previously (21). Additional amber mutants from Studier were am 290 (gene 17), am lys 3-l (gene 3.5), am 20 (gene 4), am 233 and am 111 (both in gene 6). Temperature-sensitive mutants were also obtained from F. W. Studier and included ts 106 and ts 136 (both in gene 6), ts 11 (gene 5), ts 101 (gene 4), and ts 105 (gene 19). Escherichia coli 011’ and E. coli B were the permissive and nonpermissive strains, respectively, for amber mutants (20). The following recombination-deficient hosts were used: E. coli QR 48 (recA), E. coli AB 2470 (recBBl), E. coli JC 5495 (recA13, recB21) and E. coli JC 5547 (recA13, recB21, recC22) (22). These strains were obtained from A. Becker and D. Hoar of this department. Suitably marked strains were constructed by standard phage crosses (20) and tested by complementation (20). The crosses that tested recombination profi281
Copyright All rights
0 1975 hy Academic Press, of reproduction in any form
Inc. reserved.
282
SHORT
COMMUNICATIONS
ciency were done essentially according to tage of enabling us to determine that a Studier’s method (20) with the following recombinational event yielded the two rein approximately equal modifications. The cells were grown in combinants tryptone broth which had been suppleamounts. mented with 1 mM MgSO, to aid phage We were interested in determining which adsorption. The phage and cells were T7-coded functions were involved in phage shaken manually in an adsorption tube for genetic recombination. Prior to doing this, 5 min and then centrifuged at 10,000 g for 5 it was important to establish that T7 min to remove unadsorbed phage. The titer genetic recombination did not depend on and genotype of the unadsorbed phage the major host recombination system. present in the supernatant fluid were de- From the results in Table 1, it is apparent termined. The pellet was resuspended in that T7 recombination between amber 0.8 ml of broth containing 4 mM KCN, and markers in genes 9 and 15 is not inhibited an aliquot was plated for infectious cen- by mutations in the recA, -B or -C genes. If phage recombination is someters. The remainder was diluted 5 x 105- anything, fold into broth and the final dilution was what increased in the absence of ret function. incubated at the appropriate temperature for 1 hr. The total burst was determined by We then proceeded to examine the efplating on the permissive host, E. coli 011’. fects of amber mutations in various genes upon recombination frequencies between In all crosses, the percentage of unadsorbed amber markers in genes 9 and 15. Reprewas less than 15%. The reversion frequency of single amber mutations used was less sentative results of several experiments are in Table 2. The results are than 10-4, a figure too low to influence the summarized percentage of wild-type recombinants ob- expressed as a ratio of the recombination frequency obtained for the amber mutation tained. in question over the frequency for the Recombination frequencies were determined as follows. When we wished to test amber+ allele (am/am+, final column). A decrease in this ratio in the nonpermissive the effect of a temperature-sensitive mutation on recombination between two amber host (B) would tend to implicate the mutated gene in recombination. The results markers (an, and am,), we constructed phages bearing the amber mutation and may be summarized as follows. Gene 10 the temperature-sensitive mutation in (head protein), gene 17 (tail protein), gene question (i.e., am,, ts and am,, ts). Crosses 3.5 (lysozyme) and gene 2 (reduced DNA were carried out between am, and am2 and synthesis) do not appear to be involved in genetic recombination (Expt 1-4, Table 2). between am, ts and am2 ts at temperatures ranging from 30 to 42”. The percent recomTABLE 1 binants in each case could be readily deterRECOMBINATION OF PHAGE T7 IN Ret’ AND Ret HOSTS” mined by plating for am+ recombinants on E. coli B at the permissive temperature. 5% Host Genotype Burst size Rf?COIiThis figure was doubled to account for bination reciprocal recombinants. If we wished to determine the effect of an 19.2 011’ sup- ret+ 134 amber mutation, am3, upon recombination sup ret+ 118 21.5 25.6 :R48 recA, sup 71 between am, and am, markers, we carried AB2470 recB, sup30.4 69 out a cross of the type am, am3 x am, am3 30.8 JC5495 recAB, sup+ 71 in both the permissive host (011’) and the recABC, sup+ 71 26.3 JC5547 nonpermissive host (B). In this cross, both parental phages contained two amber mua Recombination was measured between amber tations so that it was necessary to determarkers in genes 9 and 15 by measuring the percentmine the genotypes of the progeny by age of am+ recombinants among the progeny and complementation tests. This procedure, doubling this percentage to account for the reciprocal recombinants. although laborious, had the added advan-
SHORT
283
COMMUNICATIONS TABLE
2
EFFECTS OF VARIOUS AMBER MUTATIONS ON GENETIC RECOMBINATION IN PHAGE T7” Expt
Amber
Gene tested
Function affected
Host
am Burst per cell
1
13
10
Head protein
2
290
17
Tail protein
3
lys 3-1
3.5
Lysozyme
4
64
2
5
29
3
Defective DNA synthesis T7 endonuclease
6
20
4
DNA negative
7
28
5
T7 DNA polymerase
8
233
6
T7 exonuclease
I
011’ B 011’ B 011’ B 011’ B 011’ B 011’ B 011’ B 011’ B
168 2.7 131 4.8 295 11.7 305 3 280 1.7 188 1.5 298 0.93 202 1
am+
7% Recombination 24.5 15.6 27.9 21.6 13.8 14 36.3 26.2 30.6 7.2 32.2 2.8 29.6 3.5 27.5 3.8
Burst per cell 252 124 250 136 340 240 176 218 225 101 370 207 296 179 122 115
% Recombination 20 14.1 29.2 26.6 27.2 25.4 26.6 23.2 20 22.6 30.8 25.6 21.8 17.6 26.4 24.2
amlam + Relative recombination 1.2 1.1 0.96 0.81 0.51 0.55 1 1.1 1.5 0.3 1.05 0.11 1.36 0.2 1.04 0.16
” Recombination was determined between amber markers in genes 9 and 15. Results are given for the amber mutation tested (“am”) and the controls which lacked that amber mutation (“am+“).
These data show that normal recombination frequencies are obtainable even though the burst size is significantly reduced. On the other hand, mutations in genes 3-6 resulted in greatly reduced recombination when the crosses were done in a suphost (Expt 5-8, Table 2). These effects were gene specific since mutations at different sites in the same gene gave identical results. In addition, the use of urn+ revertants of the mutants tested caused recombination rates to revert to normal (data not shown). Finally, the reduced recombination effects were not dependent upon the markers between which recombination was measured in that identical results were obtained when recombination was measured between markers in genes 15 and 17 or genes 8 and 18. Having obtained suggestive evidence for the involvement of certain genes in recombination from experiments using amber mutations, we attempted to confirm these results using temperature-sensitive (ts) mutations. The ts mutation to be tested was therefore crossed into the 9am and
15am phage and the recombination frequency between these two amber markers was measured as a function of temperature. In a control experiment, a ts mutation in gene 19 (a function involved in phage DNA maturation) caused at most a limited decrease in recombination at high temperatures, while the burst size relative to the ts+ phage was reduced almost lOOO-fold (Fig. la). Temperature-sensitive mutations in gene 4 (Fig. lb), gene 5 (Fig. lc) and gene 6 (Fig. Id) caused much more marked decreases in recombination than the ts mutation in gene 19. One could argue that the effects seen using both am and ts mutations are simply due to the low burst sizes which result when the crosses are performed under nonpermissive conditions. To answer this objection, we measured genetic recombination in the presence of nalidixic acid, a drug that inhibits both phage production and DNA synthesis. As can be seen in Fig. 2, there is relatively little effect of the drug on genetic recombination, even at doses of nalidixic acid that reduce the burst sizes to the levels seen in our experiments (Table 2,
284
SHORT COMMUNICATIONS
01
001 26
30
34
38
42
Temperature
26
30
34
38
42
(‘C)
FIG. 1. Effects of ts mutations in various genes upon genetic recombination. E. coli 011’ was infected with phage bearing the ts mutations to be tested as well as amber mutations in gene 9 or 15 (the markers between which recombination is measured) at temperatures ranging from 26-42”. Recombinants were scored as am+ phage plated at room temperature. Genes tested: (a) gene 19; (b) gene 4; (c) gene 5; (d) gene 6. *-----*, Relative burst (ts/ts+); O---O, % recombination, control (ts’); O-O, % recombination, test (ts).
Fig. 1). A level of 30 pg per ml of nalidixic acid reduces the rate of T7 DNA synthesis by greater than 99% (data not shown). Thus it is unlikely that the effects of mutations in genes 3-6 are due to the nonspecific decrease in burst size. A second possibility which we cannot exclude is that DNA replication is required for recombination. The products of genes
3-6 are required for normal rates of DNA synthesis (20). If DNA replication and recombination are inseparable, then a deficiency of any of these gene products would decrease recombination rates. Although nalidixic acid abolished more than 99% of DNA synthesis without affecting recombination rates appreciably, a limited amount of DNA replication, sufficient to allow
SHORT
285
COMMUNICATIONS REFERENCES
1. STUDIER, F. W., Science 176, 367-376 (1972). 2. CENTER, M. S., STUDIER, F. W., and RICHARDSON,
C. C., hoc. Nat. Acad. Sci. USA 66, 242-248
Frc. 2. Effect of nalidixic acid on burst size and genetic recombination in phage T7. E. coli B was infected with ten phage per cell each of T7 am 9 and T7 am 15 in the presence of increasing concentrations of nalidixic acid at 30”. After 60 min. the percentage of recombinants was determined by measuring the proportion of,am+ progeny over total progeny. *-----*, burst per cell; O-0, ‘% recombination.
recombination may have occurred. Indeed, in phage X, Stahl and Stahl (23, 24) have found that recombination in most regions of the chromosome is accompanied by considerable DNA replication. These experiments suggest that some T7 gene products play a multifunctional role in DNA metabolism. T7 endonuclease I (gene 3 product) and the T7 gene 6 exonuclease have already been implicated in destruction of cellular DNA and phage DNA maturation. A recent paper has also implicated the exonuclease in genetic recombination (25). It is relatively easy to conceive of a role for an endonuclease and exonuclease in a breakage and reunion mechanism of genetic recombination (26). In addition to its role in DNA replication, the T7 DNA polymerase may contribute to the repair of recombinant molecules or its 3’ exonuclease may also be involved in trimming back redundant single strands. The role of the gene 4 product in recombination is unknown. These experiments show that at least four T7 gene products are involved in genetic recombination. The availability of all of these products in purified form (3, 5, 6, 16-18) should facilitate attempts to perform genetic recombination with T7 DNA in vitro.
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