Type II topoisomerase activities in both the G1 and G2/M phases of the dinoflagellate cell cycle

Abstract

Dinoflagellate genomes are large (up to 200 pg) and are encoded in histoneless chromosomes that are quasi-permanently condensed. This unique combination of chromosomal characteristics presents additional topological and cell cycle control problems for a eukaryotic cell, potentially exhibiting novel regulatory requirements of topoisomerase II. The heterotrophic dinoflagellate Crypthecodinium cohnii was used in this study. The topoisomerase II activities throughout its cell cycle were investigated by DNA flow cytometry following enzyme deactivation. Fluorescence microscopy was also used for studying the chromosome morphology of the treated cells. Two classes of topoisomerase II inhibitors were applied in our study, both of which caused G1 delay as well as G2/M arrest in the C. cohnii cell cycle. At high doses, the topoisomerase poisons amsacrine and ellipticine induced DNA fragmentation in C. cohnii cells. Topoisomerase II activities, as measured by the ability to decatenate kinetoplastid DNA (kDNA), are normally detected throughout the cell cycle in C. cohnii. Our results suggest that the requirement of type II topoisomerase activities during the G1 phase of the cell cycle may relate to the unwinding of quasi-permanently condensed chromosomes for the purpose of transcription. This was also the first time that topoisomerase II activity in dinoflagellate cells was detected.

Knockdown of Dinoflagellate Condensin CcSMC4 Subunit Leads to S-Phase Impediment and Decompaction of Liquid Crystalline Chromosomes

“…The degree of eventual decondensation was comparable to those observed in cation-chelated [12] or with topo II inhibitor-treated LCCs [37] ( Figure S3B) in which swelled nuclei filled the size of a normal cell. This suggested that condensins were involved in LCC compaction, likely in the restraint of proposed superhelical modules [7].…”

“…This was consistent with LCC decompaction in some ak-cells, as observed in chelation-mediated LCC decompaction in EDTA-treated nuclei [12]. As an independent control, we conducted a set of experiments with topoisomerase II inhibitor (AMSA) that induced C. cohnii LCC decondensation [37]. As in the case of some ak-cells (T = 33, transects 11-12,13-14, 19-20, and T = 70; Figure S3A), the diffused DAPI-staining (after AMSA treatment) of the whole swelled nucleus filled the whole cells ( Figure S3B), suggesting decondensed LCCs resulting from CcSMC4p knockdown.…”

“…This indicated the effect of CcSMC4p-knockdown being manifested early in the experiment and that most ak-cells had been transfected with the antisense-ODNs. Gene-knockdown would not have affected the pre-existing CcSMC4p, which in other systems have both cytoplasmic and nuclear populations [37]. Immunoblot suggested this CcSMC4p pool dropped to undetectable levels by T = 46-70 h( Figure 3B).…”

“…A flow cytogram of ak-cells exhibited sustained sub-2N populations in all later samples, contrasted with the clear gaps between G 1 -G 2 peaks peaks (yellow line, Figure 3C). There is no global decondensation during dinoflagellate DNA replication, a consequence of no normal S-phase bridge between G 1 -G 2 , and, hence, there are clear gaps between G 1 and G 2 /M flow peaks [37,38].…”

Concentration-dependent organization of DNA by the dinoflagellate histone-like protein HCc3

“…As in the case of histones, acetylation removes positive charges, thereby reducing the affinity between the histones and the DNA (79). As in the case of bacterial HU, HCc proteins may also interact functionally with topoisomerases (80–82), which are also involved in DNA replication and condensation, and is persistently expressed throughout the cell cycle of C.cohnii (83). In the presence of topoisomerase II, local topological changes of DNA filaments can possibly be modulated through the assistance of HCc proteins, in a synergistic and cooperative manner.…”