Related Experiment Videos
Determination of sequence specificity between a plasmid replication initiator protein and the origin of replication
C D Thomas1, T T Nikiforov, B A Connolly
1Department of Biochemistry, University of Leicester, UK.
Abstract:
Staphylococcal plasmids of the pT181 family replicate by a rolling circle mechanism, requiring the activities of a plasmid-specified Rep protein. The initiation event involves site-specific phosphodiester bond cleavage by Rep within the replication origin, ori. In vitro the Rep proteins also display type-I topoisomerase activity specific for this plasmid family. Although the single site of bond cleavage, ICR II, is conserved among all members of the pT181 family, the plasmid-specific Rep proteins are able to discriminate between family members in vivo, initiating replication only from the cognate origin. The basis of such specificity is believed to be due to a non-covalent binding interaction between Rep and a DNA sequence adjacent to the site of phosphodiester bond cleavage. Using the RepD protein specified by plasmid pC221, we present data for the physical parameters of RepD:oriD complex formation. Quantification of the relative strengths of the non-covalent interactions for different but related ori target sequences, measured by gel mobility shift experiments, has yielded data that are in accord with the known specificity of the protein in vivo. Oligonucleotide competition experiments demonstrate that this interaction is indeed attributable to the specificity determinant, ICR III. Protein-DNA crosslinking methods show that a carboxyl-terminal proteolytic fragment of RepD makes a specific interaction with the ICR III region of its cognate replication origin. Analysis of topoisomerase rates indicates that the interaction between ICR III and the carboxyl terminus of the protein is required before a productive interaction, namely the phosphodiester bond cleavage at the ICR II, can occur.
Insights
Staphylococcal plasmid replication relies on Rep proteins binding specifically to origins. This study details the RepD protein
Area of Science:
- Molecular Biology
- Microbial Genetics
- Biochemistry
Background:
- Staphylococcal plasmids of the pT181 family utilize a rolling circle replication mechanism.
- Replication initiation is mediated by a plasmid-encoded Rep protein that cleaves the replication origin (ori).
- Rep proteins exhibit type-I topoisomerase activity, but specifically initiate replication from cognate origins despite conserved cleavage sites (ICR II).
Purpose of the Study:
- To elucidate the physical parameters of the RepD protein:oriD complex formation.
- To understand the molecular basis of Rep protein specificity for cognate replication origins.
- To investigate the role of non-covalent interactions in replication initiation.
Main Methods:
- Gel mobility shift assays to quantify non-covalent interactions between RepD and various ori sequences.
- Oligonucleotide competition experiments to identify the specific DNA sequence involved in recognition.
- Protein-DNA crosslinking to map the interacting regions between RepD and its origin.
- Analysis of topoisomerase activity rates.
Main Results:
- Gel mobility shift data revealed quantitative differences in RepD binding affinities for related ori sequences, correlating with known in vivo specificity.
- Oligonucleotide competition confirmed that the specificity determinant is the ICR III region.
- Protein-DNA crosslinking identified a specific interaction between a RepD carboxyl-terminal fragment and the ICR III region.
- Topoisomerase activity analysis demonstrated that ICR III interaction precedes phosphodiester bond cleavage at ICR II.
Conclusions:
- The specificity of RepD binding to its cognate origin is mediated by non-covalent interactions with the ICR III region.
- A carboxyl-terminal domain of RepD is crucial for this specific recognition.
- This specific interaction is a prerequisite for the initiation of rolling circle replication by RepD.