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Light-dependent and asynchronous replication of cyanobacterial multi-copy chromosomes.
Mol Microbiol. 2012; 83:856-65
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11 Feb 2012
Confirmation, New Finding
DOI: 10.3410/f.13673957.15088057
Bacterial model organisms, namely Escherichia coli and Bacillus subtilis, played central roles in the early pioneering studies of DNA replication, and decades of research have yielded great insights into the process in those organisms. However, very little is known regarding chromosome replication and...Continue reading
Bacterial model organisms, namely Escherichia coli and Bacillus subtilis, played central roles in the early pioneering studies of DNA replication, and decades of research have yielded great insights into the process in those organisms. However, very little is known regarding chromosome replication and segregation mechanisms in other major groups of bacteria, including the cyanobacteria. Here, Watanabe et al. shed some much-needed light onto the regulation of DNA replication in polyploid cyanobacteria.
Using Synechococcus elongatus PCC 7942 as a model strain, the authors use next-generation sequencing to experimentally confirm the predicted origin of replication (ori) in growing cultures and follow the progress of its replication. Unlike in B. subtilis where a clear two-fold increase in sequencing coverage of ori/ter is observed, only a 10% difference in ori/ter is evident in S. elongatus. Instead of mapping whole-genome reads, the authors establish a method of sequencing BrdU-labeled DNA (Repi-seq) to monitor de novo DNA synthesis in cells throughout different stages of growth. This method relies on introducing a herpes simplex virus gene encoding thymidine kinase (TK) into S. elongatus and growing cells in the presence of BrdU. Through immunoprecipitation of BrdU-labeled DNA, coupled with next-generation sequencing, the authors are able to detect a clear peak around the dnaN gene. These data demonstrate that the replication origin is located upstream of the dnaN gene, and suggest that replication is initiated asynchronously among cell populations, and, furthermore, also asynchronously among the multi-copy chromosomes. This result is confirmed by quantitative real-time polymerase chain reaction (PCR) using primer sets located at different positions around the chromosome. These results also demonstrate that DNA replication proceeds bi-directionally from the origin of replication. Lastly, the authors monitor the localization of BrdU-labeled DNA using immunofluorescence microscopy, where one to two BrdU foci are typically observed. Together, the results suggest that DNA replication is initiated asynchronously amongst not only cells in the population but also different chromosomes within a cell.
Using Synechococcus elongatus PCC 7942 as a model strain, the authors use next-generation sequencing to experimentally confirm the predicted origin of replication (ori) in growing cultures and follow the progress of its replication. Unlike in B. subtilis where a clear two-fold increase in sequencing coverage of ori/ter is observed, only a 10% difference in ori/ter is evident in S. elongatus. Instead of mapping whole-genome reads, the authors establish a method of sequencing BrdU-labeled DNA (Repi-seq) to monitor de novo DNA synthesis in cells throughout different stages of growth. This method relies on introducing a herpes simplex virus gene encoding thymidine kinase (TK) into S. elongatus and growing cells in the presence of BrdU. Through immunoprecipitation of BrdU-labeled DNA, coupled with next-generation sequencing, the authors are able to detect a clear peak around the dnaN gene. These data demonstrate that the replication origin is located upstream of the dnaN gene, and suggest that replication is initiated asynchronously among cell populations, and, furthermore, also asynchronously among the multi-copy chromosomes. This result is confirmed by quantitative real-time polymerase chain reaction (PCR) using primer sets located at different positions around the chromosome. These results also demonstrate that DNA replication proceeds bi-directionally from the origin of replication. Lastly, the authors monitor the localization of BrdU-labeled DNA using immunofluorescence microscopy, where one to two BrdU foci are typically observed. Together, the results suggest that DNA replication is initiated asynchronously amongst not only cells in the population but also different chromosomes within a cell.
Disclosures
None declared
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Golden S and Cohen S: F1000Prime Recommendation of [Watanabe S et al., Mol Microbiol 2012, 83:856-65]. In F1000Prime, 11 Feb 2012; DOI: 10.3410/f.13673957.15088057. F1000Prime.com/13673957#eval15088057
F1000Prime Recommendations, Dissents and Comments for [Watanabe S et al., Mol Microbiol 2012, 83:856-65]. In F1000Prime, 18 May 2013; F1000Prime.com/13673957
