@ INSTITUTPASTEUR/EI.SEVIER Paris 1991
Res. Microbial 1991, 142, 141-149
Analysis of a copy number mutant of plasmid pSC101 : co-maintenance of wild type and mutant plasmids T. Goebel, D. Manen, C. Alff-Steinb~ger, G . X . Xia and L. Carp Department o/Molecular Biology, University o f Geneva, 30, qual Ansermet, 1211 Geneva 4 (Switzerland)
SUMMARY We have isolated a high copy number muta~ ~ of 9lasmid pSC101 which is maintained at a level 4 times higher than that of the wi~d type. The mutation is a single base change that maps in oodon 93 of the initiatiotz protein RepA, We find that the mutation relaxes the autoregulation of the protein but illcreases its affinity for the repeated sequences in the origin. The wild type and the ~,~utantrepA genes are codominant and the mutated protein ;~L',~Jn trans even in the presence of the wild type protein. Co-maintenance of the two types of plasmlds results in an intermediate copy number. Computer simulation ir,...icates that si,.m.p!emodels can explain the behaviour of the two plasmids. Key.words : Plasmid, Copy number, DNA, Replication; Mutation, PSCI01, Co-. maintenance, Codon.
INTRODUCTION In Eseherichia coil, replication of the Salmonella panama plasmid pSCI01 (Cohen and Chang, 1973; 1977) requires the plasmid-encoded 37-kDa RepA protein (Churchward et al., 1983), the host's DnaA protein (Hasunuma and Sekiguchi, 1977 ; Fray et eL, 1979; reviewed by Georgopoulos, 1989) and the integration host factor protein (IHF) (Gamas et al., 1986; Stenzel et aL, 1987, Biek and Cohen, 1989; reviewed by Friedman, 1988). The repA gene maps within a 2.2-kb HinclI-Rsal fragment which contains the essential replication region of pSCI01 (Linder et al., 1983 ; Churchward et el., 1983; Vocke and Bastia,
1983a; Yamaguchl and Yamaguchi, 1984; Armstrong et aL, 1984). In addition to the repA gene, this fragment (fig. 1) also contains the origin of replication ori, binding sites for the DnaA and IHF proteins, and par, a cis-acting locus implicated in plasmid stobility (Meacock and Cohen, 1980; Tucker et al., 1984) and copy number (Manen et al., 1990). The lHF-binding site separates a stretch of A residues from a stretch of T residues; according to Bramhill and Kornberg (1988), this asymmetric A-T rich region contains a potential entry site for the DnaB/DnaC complex. The par locus straddles a binding sitc for DNA gyrase and seems to participate in determining the state of
superhelicity of plasmid DNA (Wahle and Kornberg, 1988; Miller et al., 1990). To the left of the unique Spel site are three directly repeated sequences (RSI, RS2, RS3), 18 bp long, which are in vitro binding sites for RepA (Linder et al., 1983; Churchward et al., 1983; Vocke and Bastia, 1983a; 1983b; Armstrong et al., 1984; Yamaguchi and Yamaguchi, 1984; Sngiura et aL, 1990). A fourth partial repeat (RS4), which overlaps the Spel site, is recognized by some authors. The strongest in viiro RepA binding site, also partially homologous to the three direct repeats, is a palindromic sequence within the r e p h promoter. By binding to this palindrome, RepA blocks its
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or~ p
Res. Microbial 1991, 142, 141-149
Analysis of a copy number mutant of plasmid pSC101 : co-maintenance of wild type and mutant plasmids T. Goebel, D. Manen, C. Alff-Steinb~ger, G . X . Xia and L. Carp Department o/Molecular Biology, University o f Geneva, 30, qual Ansermet, 1211 Geneva 4 (Switzerland)
SUMMARY We have isolated a high copy number muta~ ~ of 9lasmid pSC101 which is maintained at a level 4 times higher than that of the wi~d type. The mutation is a single base change that maps in oodon 93 of the initiatiotz protein RepA, We find that the mutation relaxes the autoregulation of the protein but illcreases its affinity for the repeated sequences in the origin. The wild type and the ~,~utantrepA genes are codominant and the mutated protein ;~L',~Jn trans even in the presence of the wild type protein. Co-maintenance of the two types of plasmlds results in an intermediate copy number. Computer simulation ir,...icates that si,.m.p!emodels can explain the behaviour of the two plasmids. Key.words : Plasmid, Copy number, DNA, Replication; Mutation, PSCI01, Co-. maintenance, Codon.
INTRODUCTION In Eseherichia coil, replication of the Salmonella panama plasmid pSCI01 (Cohen and Chang, 1973; 1977) requires the plasmid-encoded 37-kDa RepA protein (Churchward et al., 1983), the host's DnaA protein (Hasunuma and Sekiguchi, 1977 ; Fray et eL, 1979; reviewed by Georgopoulos, 1989) and the integration host factor protein (IHF) (Gamas et al., 1986; Stenzel et aL, 1987, Biek and Cohen, 1989; reviewed by Friedman, 1988). The repA gene maps within a 2.2-kb HinclI-Rsal fragment which contains the essential replication region of pSCI01 (Linder et al., 1983 ; Churchward et el., 1983; Vocke and Bastia,
1983a; Yamaguchl and Yamaguchi, 1984; Armstrong et aL, 1984). In addition to the repA gene, this fragment (fig. 1) also contains the origin of replication ori, binding sites for the DnaA and IHF proteins, and par, a cis-acting locus implicated in plasmid stobility (Meacock and Cohen, 1980; Tucker et al., 1984) and copy number (Manen et al., 1990). The lHF-binding site separates a stretch of A residues from a stretch of T residues; according to Bramhill and Kornberg (1988), this asymmetric A-T rich region contains a potential entry site for the DnaB/DnaC complex. The par locus straddles a binding sitc for DNA gyrase and seems to participate in determining the state of
superhelicity of plasmid DNA (Wahle and Kornberg, 1988; Miller et al., 1990). To the left of the unique Spel site are three directly repeated sequences (RSI, RS2, RS3), 18 bp long, which are in vitro binding sites for RepA (Linder et al., 1983; Churchward et al., 1983; Vocke and Bastia, 1983a; 1983b; Armstrong et al., 1984; Yamaguchi and Yamaguchi, 1984; Sngiura et aL, 1990). A fourth partial repeat (RS4), which overlaps the Spel site, is recognized by some authors. The strongest in viiro RepA binding site, also partially homologous to the three direct repeats, is a palindromic sequence within the r e p h promoter. By binding to this palindrome, RepA blocks its
142
T. G O E B E L
E T AL.
or~ p
