JOURNAL OF VIROLOGY, Mar. 1979, p. 840-845
Vol. 29, No. 3
0022-538X/79/03-0840/06$02.00/0
Replication of Bacteriophage O>K Duplex Replicative-Form DNA in dnaB and dnaC Mutants of Escherichia coli LAWRENCE B. DUMAS* AND DANA T. BENESH Department of Biochemistry and Molecular Biology, Northwestern University, Evanston, Illinois 60201
Received for publication 4 October 1978
We have directly tested the effects of host cell DNA synthesis mutations on bacteriophage OK replicative-form (RF) DNA replication in vivo. We observed that OK RF DNA replication continued at normal rates in both dnaB and dnaC mutant hosts under conditions in which the activities of the dnaB and dnaC gene products were shown to be markedly reduced. This suggests that these two host proteins are not essential for normal OK RF DNA replication. In control experiments we observed markedly reduced rates of OK RF DNA replication in temperature-sensitive dnaG and dnaE host mutants, indicating that the products of these genes are essential. Thus, the mechanism of DNA chain initiation in vivo on the duplex RF DNA templates of isometric phages such as OK apparently is different from that on the similar templates of isometric phages such as OX174. The implications of this difference are discussed in the text.
Many small Escherichia coli isometric phages that carry single-stranded circular DNA require the host cell dnaB and dnaC gene products to replicate their duplex replicative-form (RF) DNA. These phages include the group I isometric phages 4X174, S13, and G4trl (for a review, see reference 6). In vitro experiments utilizing 4X174 singlestranded DNA as template have shown that the dnaB and dnaC proteins facilitate the synthesis of oligonucleotide primers catalyzed by the host cell dnaG protein (2, 15, 25-27). The dnaB and dnaC proteins are essential for the formation of a preinitiation complex on 4X174 template DNA, which is then recognized by the dnaG protein (15, 25, 27). These observations, together with the observed in vivo requirements for phage RF DNA replication, suggest that the group I isometric phages use the host cell dnaB and dnaC proteins in vivo to facilitate the dnaG protein catalysis of the synthesis of primer oligonucleotide chains on the replicating duplex DNA templates. Recent evidence indicates that some other small isometric E. coli phages carrying singlestranded circular DNA yield normal bursts of progeny phage upon infection of dnaB and dnaC mutant hosts at the restrictive temperature (1, 3, 12, 20, 21, 23). These phages (group II isometric phages) include St-1, 4XtB, pK, and a3 (for a review, see reference 6). However, phage production is defective in dnaG mutant hosts, indicating that the dnaG protein is required (20-22). These observations raise the possibility that group II isometric phages replicate their 840
duplex RF DNA without the need for the dnaB and dnaC host proteins; that is, DNA chain initiation could occur on these duplex DNA templates without the assistance of the dnaB and dnaC proteins. An alternative possibility that has not been excluded is that the replication of the duplex RF DNA of the group II isometric phages requires the dnaB and dnaC proteins, but RF DNA replication is not rate limiting in the production of infectious progeny phage. If true, mutations in the host dnaB and dnaC genes would show little or no effect on the synthesis of progeny phage. To determine which of these two possibilities is correct, we directly tested the effect of host cell dnaB and dnaC mutations on -OK RF DNA replication in vivo. MATERIALS AND METHODS Bacteria and phage strains. E. coli strains LD311 (uvrA- thyA- endI- dnaBt") and LD332 (uvrA- thyAendI- dnaC") are temperature-sensitive mutants of H502 (uvrA- thyA- endl) described previously (7, 14). E. coli strain C2309 (uvrA- thyA'" dnaG'") was obtained from R. Calendar. Bacteriophage
Vol. 29, No. 3
0022-538X/79/03-0840/06$02.00/0
Replication of Bacteriophage O>K Duplex Replicative-Form DNA in dnaB and dnaC Mutants of Escherichia coli LAWRENCE B. DUMAS* AND DANA T. BENESH Department of Biochemistry and Molecular Biology, Northwestern University, Evanston, Illinois 60201
Received for publication 4 October 1978
We have directly tested the effects of host cell DNA synthesis mutations on bacteriophage OK replicative-form (RF) DNA replication in vivo. We observed that OK RF DNA replication continued at normal rates in both dnaB and dnaC mutant hosts under conditions in which the activities of the dnaB and dnaC gene products were shown to be markedly reduced. This suggests that these two host proteins are not essential for normal OK RF DNA replication. In control experiments we observed markedly reduced rates of OK RF DNA replication in temperature-sensitive dnaG and dnaE host mutants, indicating that the products of these genes are essential. Thus, the mechanism of DNA chain initiation in vivo on the duplex RF DNA templates of isometric phages such as OK apparently is different from that on the similar templates of isometric phages such as OX174. The implications of this difference are discussed in the text.
Many small Escherichia coli isometric phages that carry single-stranded circular DNA require the host cell dnaB and dnaC gene products to replicate their duplex replicative-form (RF) DNA. These phages include the group I isometric phages 4X174, S13, and G4trl (for a review, see reference 6). In vitro experiments utilizing 4X174 singlestranded DNA as template have shown that the dnaB and dnaC proteins facilitate the synthesis of oligonucleotide primers catalyzed by the host cell dnaG protein (2, 15, 25-27). The dnaB and dnaC proteins are essential for the formation of a preinitiation complex on 4X174 template DNA, which is then recognized by the dnaG protein (15, 25, 27). These observations, together with the observed in vivo requirements for phage RF DNA replication, suggest that the group I isometric phages use the host cell dnaB and dnaC proteins in vivo to facilitate the dnaG protein catalysis of the synthesis of primer oligonucleotide chains on the replicating duplex DNA templates. Recent evidence indicates that some other small isometric E. coli phages carrying singlestranded circular DNA yield normal bursts of progeny phage upon infection of dnaB and dnaC mutant hosts at the restrictive temperature (1, 3, 12, 20, 21, 23). These phages (group II isometric phages) include St-1, 4XtB, pK, and a3 (for a review, see reference 6). However, phage production is defective in dnaG mutant hosts, indicating that the dnaG protein is required (20-22). These observations raise the possibility that group II isometric phages replicate their 840
duplex RF DNA without the need for the dnaB and dnaC host proteins; that is, DNA chain initiation could occur on these duplex DNA templates without the assistance of the dnaB and dnaC proteins. An alternative possibility that has not been excluded is that the replication of the duplex RF DNA of the group II isometric phages requires the dnaB and dnaC proteins, but RF DNA replication is not rate limiting in the production of infectious progeny phage. If true, mutations in the host dnaB and dnaC genes would show little or no effect on the synthesis of progeny phage. To determine which of these two possibilities is correct, we directly tested the effect of host cell dnaB and dnaC mutations on -OK RF DNA replication in vivo. MATERIALS AND METHODS Bacteria and phage strains. E. coli strains LD311 (uvrA- thyA- endI- dnaBt") and LD332 (uvrA- thyAendI- dnaC") are temperature-sensitive mutants of H502 (uvrA- thyA- endl) described previously (7, 14). E. coli strain C2309 (uvrA- thyA'" dnaG'") was obtained from R. Calendar. Bacteriophage
