Abstract:We previously mapped early-activated replication origins in the promoter regions of five abundantly transcribed genes in the slime mold Physarum polycephalum. This physical linkage between origins and genes is congruent with the preferential early replication of the active genes in mammalian cells. To determine how general this replicational organization is in the synchronous plasmodium of Physarum, we analyzed the replication of three weakly expressed genes. Bromodeoxyuridine (BrdUrd) density-shift and gene d… Show more
“…The observation of a partial Y arc when analyzing the origin-containing fragment b strongly suggests that the origin is efficiently fired. Otherwise, a complete Y arc would be observed in addition to the bubble arc, as a result of passive replication of the locus in some nuclei (34). Figure 2.Origin and replication forks mapping at php locus in early S phase.…”
Section: Resultsmentioning
confidence: 99%
“…Inactive pro A profilin gene is passively replicated in mid-S phase ( 15 , 26 ). The weakly expressed red B and red E and topoisomerase II genes are replicated early in S phase since they are embedded in a cluster of early-activated replicons [( 34 ), unpublished data]. Yet, in these cases, the genes are not coincident with an origin but with a termination site.…”
Invariance of temporal order of genome replication in eukaryotic cells and its correlation with gene activity has been well-documented. However, recent data suggest a relax control of replication timing. To evaluate replication schedule accuracy, we detailed the replicational organization of the developmentally regulated php locus that we previously found to be lately replicated, even though php gene is highly transcribed in naturally synchronous plasmodia of Physarum. Unexpectedly, bi-dimensional agarose gel electrophoreses of DNA samples prepared at specific time points of S phase showed that replication of the locus actually begins at the onset of S phase but it proceeds through the first half of S phase, so that complete replication of php-containing DNA fragments occurs in late S phase. Origin mapping located replication initiation upstream php coding region. This proximity and rapid fork progression through the coding region result in an early replication of php gene. We demonstrated that afterwards an unusually low fork rate and unidirectional fork pausing prolong complete replication of php locus, and we excluded random replication timing. Importantly, we evidenced that the origin linked to php gene in plasmodium is not fired in amoebae when php expression dramatically reduced, further illustrating replication-transcription coupling in Physarum.
“…The observation of a partial Y arc when analyzing the origin-containing fragment b strongly suggests that the origin is efficiently fired. Otherwise, a complete Y arc would be observed in addition to the bubble arc, as a result of passive replication of the locus in some nuclei (34). Figure 2.Origin and replication forks mapping at php locus in early S phase.…”
Section: Resultsmentioning
confidence: 99%
“…Inactive pro A profilin gene is passively replicated in mid-S phase ( 15 , 26 ). The weakly expressed red B and red E and topoisomerase II genes are replicated early in S phase since they are embedded in a cluster of early-activated replicons [( 34 ), unpublished data]. Yet, in these cases, the genes are not coincident with an origin but with a termination site.…”
Invariance of temporal order of genome replication in eukaryotic cells and its correlation with gene activity has been well-documented. However, recent data suggest a relax control of replication timing. To evaluate replication schedule accuracy, we detailed the replicational organization of the developmentally regulated php locus that we previously found to be lately replicated, even though php gene is highly transcribed in naturally synchronous plasmodia of Physarum. Unexpectedly, bi-dimensional agarose gel electrophoreses of DNA samples prepared at specific time points of S phase showed that replication of the locus actually begins at the onset of S phase but it proceeds through the first half of S phase, so that complete replication of php-containing DNA fragments occurs in late S phase. Origin mapping located replication initiation upstream php coding region. This proximity and rapid fork progression through the coding region result in an early replication of php gene. We demonstrated that afterwards an unusually low fork rate and unidirectional fork pausing prolong complete replication of php locus, and we excluded random replication timing. Importantly, we evidenced that the origin linked to php gene in plasmodium is not fired in amoebae when php expression dramatically reduced, further illustrating replication-transcription coupling in Physarum.
“…In this structure, the broken strand is not complementary to the strand containing the inserted ribonucleotides as deduced from resolution by in vitro branch migration. We chose to represent ribonucleotides inserted into a newly synthesized DNA strand (see "Discussion") 2001Maric et al 2002;CM, MB and GP unpublished data). To distinguish between X-shaped DNA molecules corresponding to double-Y structures and joint DNA molecules, we carried out branch migration experiments.…”
Section: Discussionmentioning
confidence: 99%
“…The redB gene is located between two replication origins firing simultaneously at the onset of S phase (Maric et al 2002). Accordingly, in Fig.…”
Section: Analysis Of Joint Dna Molecules At Replication Termination Smentioning
confidence: 99%
“…Hybridization signals were quantified by storage phosphor imaging using the Molecular Dynamics 400A and ImageQuant. We used the previously described proP, ardC (Bénard et al 2001), redB, redE (Maric et al 2002), and php (Bénard et al 2007) probes.…”
Transient four stranded joint DNA molecules bridging sister chromatids constitute an intriguing feature of replicating genomes. Here, we studied their structure and frequency of formation in Physarum polycephalum. By "3D gels", we evidenced that they are not made of four continuous DNA strands. Discontinuities, which do not interfere with the unique propensity of the joint DNA molecules to branch migrate in vitro, are linked to the crossover, enhanced by RNaseA, and affect at most half of the DNA strands. We propose a structural model of joint DNA molecules containing ribonucleotides inserted within one strand, a gapped strand, and two continuous DNA strands. We further show that spontaneous joint DNA molecules are short-lived and are as abundant as replication forks. Our results emphasize the highly frequent formation of joint DNA molecules involving newly replicated DNA in an untreated cell and uncover a transitory mechanism connecting the sister chromatids during S phase.
To determine the extent to which eukaryotic replication origins are developmentally regulated in transcriptionally competent cells, we compared origin use in untreated growing amoebae and plasmodia of Physarum polycephalum. At loci that contain genes transcribed in both developmental stages, such as the ribosomal RNA genes and two unlinked actin genes, we show that there is a similar replicational organization, with the same origins used with comparable efficiencies, as shown by twodimensional agarose-gel electrophoresis. By contrast, we found cell-type-specific replication patterns for the homologous, unlinked profilin A ( proA) and profilin P ( proP) genes. proA is replicated from a promoter-proximal origin in amoebae, in which it is highly expressed, and is replicated passively in the plasmodium, in which it is not expressed. Conversely, proP is replicated passively and is not expressed in amoebae, but coincides with an efficient origin when highly expressed in the plasmodium. Our results show a reprogramming of S phase that is linked to the reprogramming of transcription during Physarum cell differentiation. This is achieved by the use of two classes of promoter-associated replication origins: those that are constitutively active and those that are developmentally regulated. This suggests that replication origins, like genes, are under epigenetic control associated with cellular differentiation.
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