DNA replication in Physarum polycephalum: bidirectional replication of DNA within replicons

Abstract

The direction of replication of DNA within replicons of Physarum polycephalum was studied by pulse-labelling with 5-bromouracil-deoxyriboside (BrdUrd) and 3H-adenosine deoxyriboside (dAdo), followed by ultraviolet- (UV) -photolysis and analysis of molecular weights of single strand DNA fragments on alkaline sucrose gradients. Newly made DNA within replicons at all stages of completion is split in two equal halves upon UV irradiation when BrdUrd was given at the time of initiation of DNA synthesis. This shows that replication within replicons of Physarum polycephalum starts at an origin located in the center of each unit, proceeding bidirectionally from this origin.

Mapping of a Replication Origin within the Promoter Region of Two Unlinked, Abundantly Transcribed Actin Genes of Physarum polycephalum

“…Considering the known elongation rate of 0.6 kb/min per fork in the plasmodium (21,22), this finding suggests that these two genes are at most 6 kb away from a replication origin. In this study, we establish by neutral-neutral 2D gel mapping (9) and detection of nascent single-stranded replication intermediates (RIs) on alkaline gels (3) that these two abundantly transcribed actin genes are replicated at the onset of S phase from an efficient replication origin that is located within the promoter region of each gene.…”

“…The alleles of the ardB and ardC genes have identical restriction fragment length polymorphisms in strains M 3 CIV and TU 291, which are commonly selected for DNA replication studies in P. polycephalum (2,7,21,37). These two strains were used indifferently in this study.…”

Two alleles of a developmentally regulated alpha-tubulin locus in Physarum polycephalum replicate on different schedules.

“…The extended replication schedule of the altA locus seems not to be solely a matter of the larger restriction fragments with which it has been studied. Assuming a fork rate of 600 bp/min (21,22), the 7-kb altA2 fragment would be involved in replication 10 min longer than the 1-kb altB2 fragment would be. We observed, however, that the altA2 fragment changed in abundance over an interval of 25 + 5 min longer than the altB2 fragment.…”

“…The schedule of origin firing per se remains unclear for most eukaryotic genomes, since few origins have been fully characterized, and our understanding of the events that specifically define origin firing on the eukaryotic chromosome is rudimentary (reviewed in reference 33). Analysis of the initiation of S phase in P. polycephalum suggests that a single master event, in which all origins fire, occurs at the onset of S phase (21). By contrast, a late-firing origin has been described in Saccharomyces cerevisiae (19).…”

“…The average rate of replication fork movement in P. polycephalum has been estimated to be 600 bp/min (6,21,22). At this rate, a 12-to 13-kb fragment would require only about 20 min to complete replication.…”