FCCT Biochemistry Lab:Research:Plasmid Engineering:Origins of Replication
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(New page: Ori sites are locations on a genetic element (like a chromosome or a plasmid) where replication begins. There are several types of origins of replication and there seems to be some confusi...)
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Ori sites are locations on a genetic element (like a chromosome or a plasmid) where replication begins. There are several types of origins of replication and there seems to be some confusion about how they are named.
- DSO = double strand origin
- SSO = single strand origin
OriB is used in plastid (chloroplast) replication, but also in combination with specific binding proteins as an alternative origin of recplication in prokaryotes (e.g. pCU1 plasmid, where plasmid-encoded RepA is required - see the paper of Papp et al., 2000, for details).
The sinlge origin of replication on a bacterial chromosome. It has been studied in detail and it consists of two repetitive regions: three 13-mer repeats that are rich in A-T pairs is the region where strand separation occurs and four 9-mer repeats is the region where DnaA protein binds. DnaA binds to its target as an oligomer which leads to melting of A-T pairs in the other repeats. Protein DnaB (a helicase) and DnaC (an accessory protein) bind to this region and they continue to expose ssDNA for replication.
This ori site is the target site for RepA protein in R1 plasmids which initiates the replication process. For details, see the Masai and Arai 1988 paper.
This ori is linked to replication of some viruses in eukaryotic cells, but also to plasmid replication. It has been studied in detail with the pCJ1 broad host range plasmid in combination with polA-negative E. coli cells. Results have shown that oriS can act as the sole ori site, but only in presensce of RepA. (see the paper of Zoueva et al., 2003, for details).
Origin of transfer refers to the site where replication of promiscuous plasmids (mostly) by rolling circle mechanism begins. At this site one of the dsDNA strands is nicked and synthesis of a new strand begins. This new strand will be transferred as ssDNA into another cell by conjugation. Charateristic for oriT are inverted repeat sequences. These oriT sequences are binding sites for RepA proteins. As a consequence of RepA binding, a loop is formed on the DNA. The ssDNA sequence in the loop is nicked by RepA activity, followed by another nick at the nearby DSO site, to which helicase, SSBs and polymerase bind, resulting in synthesis of a complementary (leading) strand. When the whole round has been completed, RepA cleaves the displaced ssDNA product which circularizes and the complementary strand is synthesized, starting from the SSO. For details of rolling circle replication see S.A. Khan's review.
Origins of replication on plasmid backbone that are linked to "vegetative growth", which would be a counterpart of chromosomal oriC regions.