Summary
Escherichia coli rnh mutants lacking ribonuclease H (RNase H) activity can tolerate deletion of the origin of DNA Replication (ΔoriC) and transposon-insertional inactivation of an initiator gene (dnaA:Tn10). Introduction of the recA200 allele encoding a thermolabile RecA protein intornh − dnaA: Tn10 and rnh − ΔoriC mutants strains rendered DNA synthesis and colony formation of these mutants temperature sensitive. The temperature sensitivity and the broth sensitivity (Srm−) of the rnh − dnaA: Tn10 recA200 strain was suppressed by the presenceof plasmids (pBR322 derivatives) carrying dnaA +only when the intact oriC site was present on the chromosome. Lack of RNase H activity neither promoted replication of minichromosomes (pOC24 and pλasn20) in the absence of required DnaA+ protein nor inhibited dnaA +−dependent minichromosome replication. These results led to the conclusion that RNase H is not directly involved in the events leading to initiation of DNA replication at oriC. Rather, it functions as a specificity factor by eliminating certain forms of RNA-DNA hybrids which could otherwise be used to prime DNA replication at sites other than oriC.
Similar content being viewed by others
References
Atlung T (1981) Analysis of seven dnaA suppressor loci in Escherichia coli. In: Ray DL (ed) The initiation of DNA replication. ICN-UCLA Symposia on molecular and cellular biology. Academic Press, New York, pp 297–314
Bagdasarian MM, Szakowska M, Bagdasarian M (1977) Suppression of the dnaA phenotype by mutations in the rpoB cistron of ribonucleic acid polymerase in Salmonella typhimurium and Escherichia coli. J Bacteriol 130:577–582
Carl PL, Bloom L, Crouch RJ (1980) Isolation and mapping of a mutant in Escherichia coli with altered levels of ribonuclease H. J Bacteriol 144:28–35
de Massy R, Fayet O, Kogoma T (1984) Multiple origin usage for DNA replication in sdrA (rnh) mutants of Escherichia coli K-12: Initiation in the absence of oriC. J Mol Biol 178:227–236
Fuller RS, Kornberg A (1983) Purified dnaA protein in initiation of replication at the Escherichia coli chromosome origin of replication. Proc Natl Acad Sci USA 80:5817–5821
Fuller RS, Kaguni JM, Kornberg A (1981) Enzymatic replication of the origin of the Escherichia coli chromosome. Proc Natl Acad Sci USA 78:7370–7374
Fuller RS, Funnell BE, Kornberg A (1984) The dnaA protein complex with the E. coli chromosomal replication origin (oriC) and other DNA sites. Cell 38:889–900
Hansen EB, Hansen FG, Meyenburg, K von (1982) The nucleotide sequence of the dnaA gene and the first part of the dnaA gene of Escherichia coli K12. Nucleic Acids Res 10:7373–7385
Hansen EB, Atlung T, Hansen FG, Skovgaard O, Meyenburg K von (1984) Fine structure genetic map and complementation analysis of mutations in the dnaA gene of Escherichia coli. Mol Gen Genet 196:387–396
Hirose S, Hiraga S, Okazaki T (1983) Initiation site of deoxyribonucleotide polymerization at the replication origin of the Escherichia coli chromosome. Mol Gen Genet 189:422–431
Horiuchi T, Maki H, Sekiguchi M (1984) RNase H-defective mutants of Escherichia coli: a possible discriminatory role of RNase H in initiation of DNA replication. Mol Gen Genet 195:17–22
Itoh T, Tomizawa J (1980) Formation of an RNA primer for initiation of replication of ColE1 DNA by ribonuclease H. Proc Natl Acad Sci USA 77:2450–2454
Kanaya S, Crouch RJ (1984) The rnh gene is essential for growth of Escherichia coli Proc Natl Acad Sci USA 81:3447–3451
Kogoma T (1978) A novel Escherichia coli mutant capable of DNA replication in the absence of protein synthesis. J Mol Biol 121:55–69
Kogoma T (1984) The absence of RNase H allows replication of pBR322 in Escherichia coli mutants lacking DNA polymerase I. Proc Natl Acad Sci USA 81:7845–7849
Kogoma T, Meyenberg K von (1983) The origin of replication, oriC, and the dnaA protein are dispensable in stable DNA replication (sdrA) mutants of Escherichia coli K-12. EMBO J 2:463–468
Kogoma T, Subia NL (1983) polA +independent replication of concatemeric pBR322 in sdrA mutants of Escherichia coli K-12. In: Cozzarelli NR (ed) Mechanism of DNA replication and recombination, UCLA symposia on molecular and cellular biology, New Series, Alan R. Liss Inc., New York, Vol 10, pp 337–349
Kogoma T, Bialy H, Subia NL, Torrey TA, Pickett GG, Meyenburg K von (1985a) The role of RNase H in initiation of DNA replication in Escherichia coli. In: Schaechter M, Neihardt FC, Ingraham J, Kjeldgaard NO (eds) Molecular biology of bacterial growth. Jones and Bartlett Publishers, Inc, Boston (in press)
Kogoma T, Skarstad K, Boye E, Meyenburg K von, Steen HB (1985b) RecA protein acts at the initiation of stable DNA replication in rnh mutants of Escherichia coli K-12. J Bacteriol (in press)
Kornberg A (1980) DNA Replication. Freeman and Co., San Francisco
Lindahl G, Lindahl T (1984) Initiation of DNA replication in Escherichia coli: RNase H-deficient mutants do not require the dnaA function. Mol Gen Genet 196:283–289
Lloyd RG, Low B (1976) Some genetic consequences of changes in the levels of recA gene function in Escherichia coli K-12. Genetics 84:675–695
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular Cloning. Cold Spring Harbor Loboratory, Cold Spring Harbor, New York
Messer W, Meijer M, Bergmans HEN, Hansen FG, Meyenburg K von, Beck E, Schaller H (1979) Origin of replication oriC, of Escherichia coli K12 chromosome: nucleotide sequence. Cold Spring Harbor Symp Quant Biol 43:139–145
Meyenburg K von, Hansen FG (1980) The origin of replication, oriC, of the Escherichia coli chromosome: genes near oriC and construction of oriC deletion mutations. In: Alberts B, Fox CF (eds) Mechanistic studies of DNA replication and genetic recombination. ICN-UCLA symposia on molecular and cellular bilogy. Vol 19. Academic Press, New York, pp 137–159
Meyenburg K von, Hansen FG, Riise E, Bergmans HEN, Meijer M, Messer W (1979) Origin of replication, oriC, of the Escherichia coli K12 chromosome: genetic mapping and minichromosome replication. Cold Spring Harbor Symp Quant Biol 43:121–128
Meyenburg K von, Hansen FG, Atlung T, Boe L, Clausen IG, van Deurs B, Hansen EB, Jorgensen F, Koppes L, Michelsen O, Nielsen J, Pedersen PE, Rasmussen KV, Riise E, Skovgaard O (1985) Facets of the chromosomal origin of replication, oriC, of Escherichia coli. In: Schaechter M, Neihardt FC, Ingraham J, Kjeldgaad NO (eds) Molecular biology of bacterial growth, Jones and Bartlett Publishers, Inc. (in press)
Ogawa T, Pickett GG, Kogoma T, Kornberg A (1984) RNase H confers specificity in the dnaA-dependent initiation of replication at the unique origin of the Escherichia coli chromosome in vivo and in vitro. Proc Natl Acad Sci USA 81:1040–1044
Oka A, Sugimoto K, Takanami M, Hirota Y (1980) Replication origin of the Escherichia coli K-12 chromosome: the size and structure of the minimum DNA segment carrying the information for autonomous replication. Mol Gen Genet 178:9–20
Torrey TA, Kogoma T (1982) Suppressor mutations (rin) that specifically suppress the recA +dependence of stable DNA replication in Escherichia coli K-12. Mol Gen Genet 187:225–230
Torrey TA, Atlung T, Kogoma T (1984) dnaA suppressor (dasF) mutants of Escherichia coli are stable DNA replication (sdrA/rnh) mutants. Mol Gen Genet 196:350–355
Uhlin BE, Volkert MR, Clark AJ, Sancar A, Rupp WP (1982) Nucleotide sequence of a recA operator mutation. Mol Gen Genet 185:251–254
Author information
Authors and Affiliations
Additional information
Communicated by H. Saedler
Rights and permissions
About this article
Cite this article
Kogoma, T., Subia, N.L. & von Meyenburg, K. Function of ribonuclease H in initiation of DNA replication in Escherichia coli K-12. Molec. Gen. Genet. 200, 103–109 (1985). https://doi.org/10.1007/BF00383320
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00383320