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Rolling-circle plasmid pKYM re-initiates DNA replication
1Division of Life Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Japan.
Abstract:
It is believed that rolling-circle plasmids are incapable of re-initiation since they have to maintain their copy number and this is one of the differences between plasmids and phages as phi-x174. To examine whether a rolling-circle plasmid pKYM is incapable of re-initiating DNA replication, we constructed a plasmid that carries both the pKYM origin (fragment 13, 173 bp) and its truncated origin (fragment 32, 56 bp) in the same orientation. This plasmid yielded two smaller plasmids in the presence of RepK, an initiator protein. We showed that RepK can bind to the fragment 13 but not to fragment 32 which lacks the 3'- moiety of fragment 13. These results imply that RepK initiates DNA replication from fragment 13 and terminates at fragment 32, then the same RepK is used for re-initiation of replication from the fragment 32 region. pKYM is likely to be a unique plasmid that re-initiates DNA replication like a phase phi-x174.
Insights
Rolling-circle plasmid pKYM demonstrates re-initiation of DNA replication, challenging previous beliefs. This unique plasmid utilizes the initiator protein RepK for both initiation and re-initiation, similar to bacteriophage phi-X174.
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- Rolling-circle replication is a mechanism used by plasmids and viruses for DNA synthesis.
- Previously, rolling-circle plasmids were thought to be incapable of re-initiating replication to maintain copy number, unlike phages such as phi-X174.
Purpose of the Study:
- To investigate whether the rolling-circle plasmid pKYM can re-initiate DNA replication.
- To understand the role of the initiator protein RepK in pKYM replication.
Main Methods:
- Constructed a plasmid containing both the pKYM origin (fragment 13) and a truncated origin (fragment 32) in the same orientation.
- Assessed the binding of RepK to different origin fragments.
- Analyzed plasmid products generated in the presence of RepK.
Main Results:
- The constructed plasmid yielded two smaller plasmids in the presence of RepK.
- RepK bound to fragment 13 but not to fragment 32.
- Replication initiated at fragment 13 and terminated at fragment 32, with subsequent re-initiation from the fragment 32 region by RepK.
Conclusions:
- The rolling-circle plasmid pKYM is capable of re-initiating DNA replication.
- RepK plays a crucial role in both initiation and re-initiation of pKYM replication.
- pKYM exhibits a unique replication strategy, similar to bacteriophage phi-X174, by re-initiating DNA replication.