Summary
Recombinant DNA techniques were used to study varibus aspects of rep gene function in Escherichia coli. In order to enhance expression of the Rep protein, the rep gene was cloned into the vector pKC30 under the control of the λp L promoter. By trimming away a portion of the DNA sequence immediately upstream of the translational start site of rep, we were able to obtain very high levels of Rep protein upon induction. Cells carrying such plasmids showed no ill effects from the high concentration of the protein. To ascertain the consequence of the absence of Rep protein on the cell, the chromosomal copy of the gene was deleted using a homologous recombination technique. The viability of E. coli strains completely lacking the rep gene proves that the Rep function is not essential, at least in wild-type cells under laboratory conditions. We confirmed that in the absence of Rep function there is an increase in the average number of growing forks in exponentially growing cells; augmentation of Rep protein levels above normal, however, did not detectably decrease the number of growing forks.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- RBS:
-
ribosome binding site
- kan:
-
kanamycin
- Ap:
-
ampicillin
- Tc:
-
tetracycline
- CAM:
-
chloramphenicol
References
Bialkowska-Hobrzanska H, Denhardt DT (1984) Cloning and analysis of the functional rep gene of E. coli K12. Gene 28:93–102
Bialkowska-Hobrzanska H, Gilchrist CA, Denhardt DT (1985) Identification of the promoter and N-terminus of the Rep protein. J Bacteriol 164:1004–1010
Bridges BA, von Wright A (1981) Influence of mutations at the rep gene on survival of Escherichia coli following ultraviolet light irradiation or 8-methoxypsoralen photosensitization. Evidence for a recA + rep +-dependent pathway for repair of DNA crosslinks. Mutat Res 82:229–238
Colasanti J, Denhardt DT (1985) Expression of the cloned ϕX174 A* gene in E. coli inhibits DNA replication and cell division. J Virol 53:807–813
Cooper S, Weusthoff G (1971) Comment on the use of chloramphenicol to study the initiation of deoxyribonucleic acid synthesis. J Bacteriol 106:709–711
Davis RW, Botstein D, Roth JR (eds) (1980) Advanced bacterial genetics. Cold Spring Harbor Laboratory, New York
Denhardt DT, Iwaya M, Larison LL (1972) The rep mutation II. Its effect on Escherichia coli and on the replication of bacteriophage ϕX174. Virology 49:486–496
Dennis PP, Nomura M (1975) Regulation of expression of the ribosomal protein genes of E. coli. J Mol Biol 97:61–76
Evans IM, Eberle H (1975) Accumulation of the capacity for initiation of deoxyribonucleic acid replication in Escherichia coli. J Bacteriol 121:883–891
Gilchrist C, Denhardt DT (1987) Escherichia coli rep gene: Sequence of the gene, the encoded helicase, and its homology with uvrD. Nucleic Acids Res 15:465–475
Gribskow M, Burgess RR (1983) Overexpression and purification of the sigma subunit of E. coli RNA polymerase. Gene 26:109–118
Joyce CM, Grindley NDF (1983) Construction of a plasmid that overproduces the large proteolytic fragment (Klenow) of DNA polymerase I of E. coli. Proc Natl Acad Sci USA 80:1830–1834
Joyce CM, Grindley NDF (1984) Method for determining whether a gene of E. coli is essential: Application to the polA gene. J Bacteriol 158:643–676
Kahn M, Kolter R, Thomas C, Figursky D, Meyer R, Remant D, Helinski DR (1979) Plasmid cloning vehicles derived from plasmids ColE1, F, R6K and RK2. Methods Enzymol 68:227–280
Kornberg A, Scott JF, Bertsch L (1978) ATP utilization by rep protein in the catalytic separation of DNA strands at a replicating fork. J Biol Chem 253:3298–3304
Lane HED, Denhardt DT (1975) The rep mutation IV: Slower movement of replicating forks in E. coli rep strains. J Mol Biol 97:99–112
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: A laboratory manual. Cold Spring Laboratory, New York
Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, New York
Olins PO, Erickson BD, Burgess RR (1983) Overproduction of E. coli Nus A protein. Gene 26:11–18
Queen C (1983) A vector that uses phage signals for efficient synthesis of proteins in Escherichia coli. J Mol Appl Genet 2:1–10
Rosenberg M, Ho Y, Shatzman A (1981) The use of pKC30 and its derivatives for controlled expression of genes. Methods Enzymol 101:123–142
Schleif RF, Wensink PC (1981) Practical methods in molecular biology. Springer-Verlag, New York, pp 27–41
Scott JF, Kornberg A (1978) Purification of the Rep protein of E. coli. J Biol Chem 253:3292–3297
Scott JF, Eisenberg S, Bertsch LL, Kornberg A (1977) A mechanism of duplex DNA replication revealed by enzymatic studies of phage ϕX174: Catalytic strand separation in advance of replication. Proc Natl Acad Sci USA 74:193–197
Shimatake H, Rosenberg M (1981) Purified λ regulatory protein cII positively activates promoters for lysogenic development. Nature 292:128–132
Takahashi S, Hours C, Iwaya M, Lane HED, Denhardt DT (1978) In: Denhardt DT, Dressler DH, Ray DS (eds) The single-stranded DNA phages. Cold Spring Harbor Laboratory, New York, pp 393–400
Takahashi S, Hours C, Chu A, Denhardt DT (1979) The rep mutation. VI. Purification and properties of the Escherichia coli rep protein, DNA helicase III. Can J Biochem 57:855–866
Taucher-Scholz G, Abdel-Monem M, Hoffmann-Berling H (1983) In: Cozzarelli NR (ed) Mechanisms of DNA replication and recombination. Alan R. Liss, Inc., New York, pp 65–76
Yarranton GT, Gefter ML (1979) Enzyme-catalyzed DNA unwinding: Studies on Escherichia coli rep protein. Proc Natl Acad Sci USA 76:1658–1662
Yasuda S, Hirota Y (1977) Cloning and mapping of the replication origin of E. coli. Proc Natl Acad Sci USA 74:5458–5462
Zettlmeissl G, Kaczorek M, Moya M, Streeck RE (1986) Expression of immunologically reactive diphtheria toxin fusion proteins under the control of the pR promoter of λ. Gene 41:102–111
Zuker M, Stiegler P (1981) Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acids Res 9:133–148
Author information
Authors and Affiliations
Additional information
Communicated by N.D.F. Grindley
The previous paper in this series is Gilchrist and Denhardt (1987)
Rights and permissions
About this article
Cite this article
Colasanti, J., Denhardt, D.T. The Escherichia coli rep mutation. X. Consequences of increased and decreased Rep protein levels. Molec Gen Genet 209, 382–390 (1987). https://doi.org/10.1007/BF00329669
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00329669