ena-STUDY-Centre for Systems and Synthetic Biology-17-10-2014-12:10:43:606-395
The consequences of replicating in the wrong orientation: bacterial chromosome duplication without an active replication origin
Chromosome duplication is normally strictly regulated in all organisms via the assembly step of replication fork complexes at specific replication origins. In Escherichia coli the DnaA replication initiator protein regulates the recruitment of the replication machinery to the single origin of replication, oriC. This regulation can be undermined by certain defects in nucleic acid metabolism. A prominent example are cells lacking RNase HI, in which replication initiates independently of both DnaA and oriC, presumably at persisting R-loops. A similar mechanism was assumed for origin-independent synthesis observed in cells lacking RecG. However, we postulated recently that this synthesis initiates at intermediates resulting from the collision of two replication forks in the termination area of the chromosome. We have now further analysed the roles of RecG and RNase HI in vivo. We show that the genetic requirements for origin-independent synthesis differ significantly in cells lacking RecG and RNase HI, in line with the idea that synthesis arises from two very different mechanisms. However, regardless of how synthesis is initiated, a portion of forks starting away from oriC will proceed in an orientation opposite to normal replication. We show that the resulting problems, such as head-on encounters with ongoing transcription, are severe enough to threaten the viability of the cells affected. Our data suggest that RecG and RNase HI, despite their different functions, are important factors for maintaining control of replication initiation and replication orientation. The absence of either protein leads to serious problems to chromosome replication, thereby highlighting the advantages of the normal chromosome arrangement that exploits a single origin, thereby controlling the number of forks and their orientation in relation to transcription, as well as a defined termination area for the safe processing of fork collision intermediates. Changes to this arrangement cause problems to cell cycle control, chromosome dynamics and, ultimately, cell viability.
Chromosome duplication is normally strictly regulated in all organisms via the assembly step of replication fork complexes at specific replication origins. In Escherichia coli the DnaA replication initiator protein regulates the recruitment of the replication machinery to the single origin of replication, oriC. This regulation can be undermined by certain defects in nucleic acid metabolism. A prominent example are cells lacking RNase HI, in which replication initiates independently of both DnaA and oriC, presumably at persisting R-loops. A similar mechanism was assumed for origin-independent synthesis observed in cells lacking RecG. However, we postulated recently that this synthesis initiates at intermediates resulting from the collision of two replication forks in the termination area of the chromosome. We have now further analysed the roles of RecG and RNase HI in vivo. We show that the genetic requirements for origin-independent synthesis differ significantly in cells lacking RecG and RNase HI, in line with the idea that synthesis arises from two very different mechanisms. However, regardless of how synthesis is initiated, a portion of forks starting away from oriC will proceed in an orientation opposite to normal replication. We show that the resulting problems, such as head-on encounters with ongoing transcription, are severe enough to threaten the viability of the cells affected. Our data suggest that RecG and RNase HI, despite their different functions, are important factors for maintaining control of replication initiation and replication orientation. The absence of either protein leads to serious problems to chromosome replication, thereby highlighting the advantages of the normal chromosome arrangement that exploits a single origin, thereby controlling the number of forks and their orientation in relation to transcription, as well as a defined termination area for the safe processing of fork collision intermediates. Changes to this arrangement cause problems to cell cycle control, chromosome dynamics and, ultimately, cell viability.