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Mutations in direct repeat sequences and in a conserved sequence adjacent to the repeats result in a defective
Abstract:
Plasmid pMM3 is a pBR322 derivative carrying the gamma origin of replication of the naturally occurring plasmid R6K. We have produced a gamma-origin mutant bank of this plasmid using the single-strand-specific mutagen sodium bisulfite. Members of this bank contain single or multiple mutations in the seven direct repeats and the flanking sequences in the gamma origin. Three mutants with defective gamma origins have been isolated from this mutant bank. Two of these direct repeat mutants, gamma 117 and gamma 120, are unable to replicate and also have lost the ability to bind the R6K initiation protein pi in vitro at one of the seven 22-base-pair direct repeats within their respective origins. Precise deletion of the damaged repeat of either of these mutants restores origin function, suggesting that the primary defect of these mutants involves a disruption of the normal spacing of pi binding and flanking sequences within the gamma origin. The third mutant, gamma 111, binds pi normally but replicates at a greatly reduced copy number due to a mutation near the seventh repeat. This mutation falls within a short sequence that appears to be conserved among a number of other plasmids that contain direct repeats within their origins of replication.
Insights
Mutations in the R6K gamma origin of replication disrupt plasmid replication and protein binding. Restoring repeat sequences or altering flanking regions can restore origin function, highlighting the importance of precise DNA spacing for replication initiation.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- The R6K plasmid's gamma origin of replication is crucial for its replication control.
- Understanding the R6K gamma origin's functional elements is key to plasmid biology.
Purpose of the Study:
- To investigate the role of direct repeats and flanking sequences within the R6K gamma origin of replication.
- To identify specific mutations affecting gamma origin function and replication initiation.
Main Methods:
- Generation of a gamma-origin mutant bank using sodium bisulfite mutagenesis.
- Isolation and characterization of mutant plasmids with defective gamma origins.
- In vitro binding assays with the R6K initiation protein pi.
Main Results:
- Two mutants (gamma 117, gamma 120) showed defective replication and impaired pi protein binding due to mutations in direct repeats.
- Precise deletion of mutated repeats restored origin function in gamma 117 and gamma 120.
- A third mutant (gamma 111) exhibited normal pi binding but reduced replication copy number due to a mutation near the seventh repeat.
Conclusions:
- The spacing and integrity of direct repeats and flanking sequences are critical for R6K gamma origin function.
- Mutations disrupting pi binding sites or their spacing significantly impair replication.
- A conserved sequence near the seventh repeat may play a role in replication control.