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Replication origin mutations affecting binding of pSC101 plasmid-encoded Rep initiator protein
A Arini1, M Tuscan, G Churchward
1Department of Microbiology and Immunology, Emory University, Atlanta, Georgia 30322.
Abstract:
To investigate the role of binding sites for Rep initiation protein in the replication of pSC101, a series of plasmids was constructed which carried different combinations of mutations in three binding sites within the minimal origin of replication. Mutation of all three sites reduced the affinity of purified Rep protein for the origin by 100-fold, as measured by a competition binding assay. Mutations in individual binding sites prevented binding of Rep protein to the mutant site but not to adjacent wild-type sites. Transformation efficiency, copy number, and stability over 150 generations were measured for each of the mutant plasmids. Unlike other similar plasmids related to pSC101, the Rep binding sites were found not to be equivalent. A mutation in the site RS1, proximal to repeated sequences which serve as DnaB helicase entry sites in oriC, had a severe effect on replication activity. A similar mutation in the distal site RS3 caused a reduction in copy number, but the mutant plasmid was stably maintained despite a broadened distribution of copy number within the population. A mutation in the middle RS2 site had no significant effect on pSC101 replication.
Insights
Investigating Rep protein binding sites in pSC101 plasmid replication revealed non-equivalent sites. Mutations in RS1 severely impacted replication, while RS3 mutations reduced copy number but maintained stability.
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- The pSC101 plasmid is a low-copy-number bacterial plasmid crucial for molecular cloning.
- Replication initiation of pSC101 depends on the Rep initiation protein binding to specific sites within the origin of replication.
Purpose of the Study:
- To elucidate the functional significance of individual Rep protein binding sites within the pSC101 minimal origin of replication.
- To determine the impact of mutations in these binding sites on plasmid replication, copy number, and stability.
Main Methods:
- Construction of pSC101 plasmids with various mutations in the three Rep binding sites (RS1, RS2, RS3).
- Assessing Rep protein affinity for the origin using competition binding assays.
- Measuring transformation efficiency, plasmid copy number, and stability over 150 generations for mutant plasmids.
Main Results:
- Mutating all three Rep binding sites reduced Rep protein affinity by 100-fold.
- Individual site mutations differentially affected Rep binding and plasmid replication.
- Mutation in RS1 (proximal to DnaB helicase entry sites) severely impaired replication.
- Mutation in RS3 (distal site) reduced copy number but maintained plasmid stability.
- Mutation in RS2 (middle site) had no significant effect on pSC101 replication.
Conclusions:
- The Rep binding sites within the pSC101 origin are not functionally equivalent.
- The RS1 binding site plays a critical role in pSC101 replication initiation and efficiency.
- Plasmid stability can be maintained even with reduced copy number and broader copy number distribution, depending on the specific mutation.