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Replication control and incompatibility in bacterial plasmids
198 2 Buhrow, S. A., Cohen, S. and Staros, J. V. (in press) 3 Cohen, S., Carpenter, G. and King, L., Jr (1980) J. BioL (hem. 255, 4834-4842 4 Linsley, P. S. and Fox, C. F. (1980)J. Supramol. Stract. 14, 461-471 5 Schreiber, A. B.. Yardin, Y. and Schlessinger, J. (1981) Biochem. Biophys. Res. ('ommun. 101.
517-523
]rIBS - J u n e 1982 6 Schlessinger, J., Schecter, Y., Willingham, M. C. and Pastan, 1. (1978)Proc. NatlAcad. Sci. U.S.A. 75, 2659-2663 7 Schlechter, Y., Hernaez. L., Schlessinger. J. and Cuatrecasas, (1979) Nature (London) 278, 835-837 8 Richards, F. M. and Vithayathil, P. J. (1959) J. BioL Chem. 234, 1459-1465 9 Frey. P., Forand, R., Maciag. T. and Shooter,
Replication control and incompatibility in bacterial plasmids Bacterial plasmids are one of the most attractive model systems for studying the control of DNA replication. Plasmids replicate in a controlled manner in harmony with the cell cycle so that the number of copies per cell is maintained. A property which is related to the mechanism of replication control is that of 'incompatibility', whereby two related plasmids are unable to coexist in the same cell. The currently favoured model is essentially that of Pritchard et al., 1 which states that replication is controlled by a repressor substance, the specific activity of which is linked to cell growth. When the repressor activity falls below a threshold level, replication commences and simultaneously more repressor is produced preventing further cycles of replication. Two incompatible plasmids have identical repressors and repressor target sites. Studies using plasmid ColE1 have given some insight as to how copy number control and incompatibility may operate. Two species of RNA are encoded in the region of the replication origin. One species, RNA II is transcribed from a point upstream from and through the origin, terminating beyond it. RNase H processes RNA II at the origin to form a primer upon which DNA polymerase I acts to initiate replication. RNA I is a smaller species which is transcribed in the opposite direction to RNA II, starting at a region known to be involved in copy number control. The coding sequences for RNA I and RNA II overlap and are thus complementary. RNA I prevents processing of RNA II by RNase H. The mechanism by which RNA I acts has been investigated by Lacatena and Cesareni2 who isolated mutants insensitive to the ColE1 repressor. Repressorinsensitive mutants are presumed to be
altered in the repressor target site. In theory, such mutants should lack copy number control and exist at a higher than normal copy number; indeed three isolates, svir2, svir7 and svir12 do. However, one isolate, s v i r l l , exists at a lower copy number. Preliminary experiments with the mutant plasmids3 suggested that the repressor target site overlapped with the repressor coding sequences. Thus a mutation in the target site should also result in an altered repressor. Do the mutant plasmids still have active repressors, as appears to be the case with s v i r l l ? Derivatives were made of the four isolates so that each derivative could be clearly distinguished from its parent. Each derivative was then used to superinfect a strain carrying one of the mutant parents. All four parent plasmids Were able to repress the replication of their respective derivatives. None of the plasmids except svir2 andsvir7 could repress the replication of wild-type ColE1 or those derived from other mutant isolates, svir2 and svir7 only cross react with each other and appear to be identical. Therefore, all the mutant plasmids still produce active repressors, The repressor target sites and their respective repressors are altered in a complementary manner. The mutant repressors can no longer recognize wild-type target sites or even other altered target sites. This lack of recognition between different isolates and wild-type ColE1 enables the mutant plasmids to coexist with one another, but still be incompatible with any plasmid that is derived from them, i.e. the mutations have created new compatibility groups. If each type of plasmid still has an active repressor and target site then why do three of the isolates exist at a higher than normal copy
~ElsevierBiomedicalPress 1982 0376 5067/82/0000- t,~00/$027,
E. M. (1979) Proc. Nad Acad. Sci. U.S.A. 76, 6294-.6298 T. MACIAG
Department of Pathology, Harvard Medical Scht~l, Charles A. Dana Research Institute, Beth Israel Hospital, Boston. MA 02215, U.S.A.
number? Sequencing of the mutations shows that they occur in a region of RNA I and II that has significant secondary structure. RNA I can probably form a complex structure closely resembling tRNA. All four of the mutations fall in the region analogous to the anticodon. If RNA I is the replication inhibitor, which Seems likely, then its complementary sequence in RNA I1 is the repressor target site. This study suggests that it is the bases in the 'anticodon' loop of RNA I that are primarily involved in replication inhibition. The three high copy number isolates change in RNA I a C base to a U, making a weaker A - U base pair between the repressor and target site. The low copy number isolate, s v i r l l , changes an A to a G making a more effective G--C base pair. These findings explain how repressor and target site are altered in a complementary manner and how compatibility groupings are determined by the three base code in the 'anticodon'. Recent evidence suggests that this model may not just apply to ColE l-like plasmids. Similar complementary RNAs have been identified near the origin of replication of plasmids RI and R6-5 which are related to the E. coli F sex factoP .~. References I Pritchard, R. H., Barth, P. T. and Collins J. (I969) Syrup. Soc. Gen. Mierobiol. 19, 293-298 2 Laeatena, R. M. and Cesareni. G. (1981)Nature (London) 245. 623-626 3 Cesareni, G. ( 1981 ) Molec. Gen. Genet. 184, 4245 4 Danbara, H., Brady, G., Timmis, J. K. and Timmis, K. N. (198l) Proc. Natl Acad. Sci. U.S.A. 78, 4699-4703 5 Stougaard, P., Molin, S. and Nordstrom, K. ( 1981 ) Proc. Natl Acad. Sci. U.S.A. 78, 6008--.(~12
MARTIN WATSON Department of Botany, University of Durham, South Road, Durham DHI 3LE, U.K.
517-523
]rIBS - J u n e 1982 6 Schlessinger, J., Schecter, Y., Willingham, M. C. and Pastan, 1. (1978)Proc. NatlAcad. Sci. U.S.A. 75, 2659-2663 7 Schlechter, Y., Hernaez. L., Schlessinger. J. and Cuatrecasas, (1979) Nature (London) 278, 835-837 8 Richards, F. M. and Vithayathil, P. J. (1959) J. BioL Chem. 234, 1459-1465 9 Frey. P., Forand, R., Maciag. T. and Shooter,
Replication control and incompatibility in bacterial plasmids Bacterial plasmids are one of the most attractive model systems for studying the control of DNA replication. Plasmids replicate in a controlled manner in harmony with the cell cycle so that the number of copies per cell is maintained. A property which is related to the mechanism of replication control is that of 'incompatibility', whereby two related plasmids are unable to coexist in the same cell. The currently favoured model is essentially that of Pritchard et al., 1 which states that replication is controlled by a repressor substance, the specific activity of which is linked to cell growth. When the repressor activity falls below a threshold level, replication commences and simultaneously more repressor is produced preventing further cycles of replication. Two incompatible plasmids have identical repressors and repressor target sites. Studies using plasmid ColE1 have given some insight as to how copy number control and incompatibility may operate. Two species of RNA are encoded in the region of the replication origin. One species, RNA II is transcribed from a point upstream from and through the origin, terminating beyond it. RNase H processes RNA II at the origin to form a primer upon which DNA polymerase I acts to initiate replication. RNA I is a smaller species which is transcribed in the opposite direction to RNA II, starting at a region known to be involved in copy number control. The coding sequences for RNA I and RNA II overlap and are thus complementary. RNA I prevents processing of RNA II by RNase H. The mechanism by which RNA I acts has been investigated by Lacatena and Cesareni2 who isolated mutants insensitive to the ColE1 repressor. Repressorinsensitive mutants are presumed to be
altered in the repressor target site. In theory, such mutants should lack copy number control and exist at a higher than normal copy number; indeed three isolates, svir2, svir7 and svir12 do. However, one isolate, s v i r l l , exists at a lower copy number. Preliminary experiments with the mutant plasmids3 suggested that the repressor target site overlapped with the repressor coding sequences. Thus a mutation in the target site should also result in an altered repressor. Do the mutant plasmids still have active repressors, as appears to be the case with s v i r l l ? Derivatives were made of the four isolates so that each derivative could be clearly distinguished from its parent. Each derivative was then used to superinfect a strain carrying one of the mutant parents. All four parent plasmids Were able to repress the replication of their respective derivatives. None of the plasmids except svir2 andsvir7 could repress the replication of wild-type ColE1 or those derived from other mutant isolates, svir2 and svir7 only cross react with each other and appear to be identical. Therefore, all the mutant plasmids still produce active repressors, The repressor target sites and their respective repressors are altered in a complementary manner. The mutant repressors can no longer recognize wild-type target sites or even other altered target sites. This lack of recognition between different isolates and wild-type ColE1 enables the mutant plasmids to coexist with one another, but still be incompatible with any plasmid that is derived from them, i.e. the mutations have created new compatibility groups. If each type of plasmid still has an active repressor and target site then why do three of the isolates exist at a higher than normal copy
~ElsevierBiomedicalPress 1982 0376 5067/82/0000- t,~00/$027,
E. M. (1979) Proc. Nad Acad. Sci. U.S.A. 76, 6294-.6298 T. MACIAG
Department of Pathology, Harvard Medical Scht~l, Charles A. Dana Research Institute, Beth Israel Hospital, Boston. MA 02215, U.S.A.
number? Sequencing of the mutations shows that they occur in a region of RNA I and II that has significant secondary structure. RNA I can probably form a complex structure closely resembling tRNA. All four of the mutations fall in the region analogous to the anticodon. If RNA I is the replication inhibitor, which Seems likely, then its complementary sequence in RNA I1 is the repressor target site. This study suggests that it is the bases in the 'anticodon' loop of RNA I that are primarily involved in replication inhibition. The three high copy number isolates change in RNA I a C base to a U, making a weaker A - U base pair between the repressor and target site. The low copy number isolate, s v i r l l , changes an A to a G making a more effective G--C base pair. These findings explain how repressor and target site are altered in a complementary manner and how compatibility groupings are determined by the three base code in the 'anticodon'. Recent evidence suggests that this model may not just apply to ColE l-like plasmids. Similar complementary RNAs have been identified near the origin of replication of plasmids RI and R6-5 which are related to the E. coli F sex factoP .~. References I Pritchard, R. H., Barth, P. T. and Collins J. (I969) Syrup. Soc. Gen. Mierobiol. 19, 293-298 2 Laeatena, R. M. and Cesareni. G. (1981)Nature (London) 245. 623-626 3 Cesareni, G. ( 1981 ) Molec. Gen. Genet. 184, 4245 4 Danbara, H., Brady, G., Timmis, J. K. and Timmis, K. N. (198l) Proc. Natl Acad. Sci. U.S.A. 78, 4699-4703 5 Stougaard, P., Molin, S. and Nordstrom, K. ( 1981 ) Proc. Natl Acad. Sci. U.S.A. 78, 6008--.(~12
MARTIN WATSON Department of Botany, University of Durham, South Road, Durham DHI 3LE, U.K.