Sue Cotterill

Depletion of Drad21/Scc1 in Drosophila Cells Leads to Instability of the Cohesin Complex and Disruption of Mitotic Progression

BACKGROUND The coordination of cell cycle events is necessary to ensure the proper duplication and dissemination of the genome. In this study, we examine the consequences of depleting Drad21 and SA, two non-SMC subunits of the cohesin complex, by dsRNA-mediated interference in Drosophila cultured cells. RESULTS We have shown that a bona fide cohesin complex exists in Drosophila embryos. Strikingly, the Drad21/Scc1 and SA/Scc3 non-SMC subunits associate more intimately with one another than they do with the SMCs. We have observed defects in mitotic progression in cells from which Drad21 has been depleted: cells delay in prometaphase with normally condensed, but prematurely separated, sister chromatids and with abnormal spindle morphology. Much milder defects are observed when SA is depleted from cells. The dynamics of the chromosome passenger protein, INCENP, are affected after Drad21 depletion. We have also made the surprising observation that SA is unstable in the absence of Drad21; however, we have shown that the converse is not true. Interference with Drad21 in living Drosophila embryos also has deleterious effects on mitotic progression. CONCLUSIONS We conclude that Drad21, as a member of a cohesin complex, is required in Drosophila cultured cells and embryos for proper mitotic progression. The protein is required in cultured cells for chromosome cohesion, spindle morphology, dynamics of a chromosome passenger protein, and stability of the cohesin complex, but apparently not for normal chromosome condensation. The observation of SA instability in the absence of Drad21 implies that the expression of cohesin subunits and assembly of the cohesin complex will be tightly regulated.

Enigmatic variations: divergent modes of regulating eukaryotic DNA replication.

Proteins involved in DNA replication are conserved from yeast to mammals, suggesting that the mechanism was established at an early stage of eukaryotic evolution. In spite of this common origin, recent findings have revealed surprising variations in how replication initiation is controlled, implying that a conserved mechanism has not necessarily resulted in regulatory conservation.

Drosophila CAP-D2 is required for condensin complex stability and resolution of sister chromatids

The precise mechanism of chromosome condensation and decondensation remains a mystery, despite progress over the last 20 years aimed at identifying components essential to the mitotic compaction of the genome. In this study, we analyse the localization and role of the CAP-D2 non-SMC condensin subunit and its effect on the stability of the condensin complex. We demonstrate that a condensin complex exists in Drosophila embryos, containing CAP-D2, the anticipated SMC2 and SMC4 proteins, the CAP-H/Barren and CAP-G (non-SMC) subunits. We show that CAP-D2 is a nuclear protein throughout interphase, increasing in level during S phase, present on chromosome axes in mitosis, and still present on chromosomes as they start to decondense late in mitosis. We analysed the consequences of CAP-D2 loss after dsRNA-mediated interference, and discovered that the protein is essential for chromosome arm and centromere resolution. The loss of CAP-D2 after RNAi has additional downstream consequences on the stability of CAP-H, the localization of DNA topoisomerase II and other condensin subunits, and chromosome segregation. Finally, we discovered that even after interfering with two components important for chromosome architecture (DNA topoisomerase II and condensin), chromosomes were still able to compact, paving the way for the identification of further components or activities required for this essential process.

Differential Requirements for MCM Proteins in DNA Replication in Drosophila S2 Cells

Background The MCM2-7 proteins are crucial components of the pre replication complex (preRC) in eukaryotes. Since they are significantly more abundant than other preRC components, we were interested in determining whether the entire cellular content was necessary for DNA replication in vivo. Methodology/Principle Findings We performed a systematic depletion of the MCM proteins in Drosophila S2 cells using dsRNA-interference. Reducing MCM2-6 levels by >95–99% had no significant effect on cell cycle distribution or viability. Depletion of MCM7 however caused an S-phase arrest. MCM2-7 depletion produced no change in the number of replication forks as measured by PCNA loading. We also depleted MCM8. This caused a 30% reduction in fork number, but no significant effect on cell cycle distribution or viability. No additive effects were observed by co-depleting MCM8 and MCM5. Conclusions/Significance These studies suggest that, in agreement with what has previously been observed for Xenopus in vitro, not all of the cellular content of MCM2-6 proteins is needed for normal cell cycling. They also reveal an unexpected unique role for MCM7. Finally they suggest that MCM8 has a role in DNA replication in S2 cells.

GINS Inactivation Phenotypes Reveal Two Pathways for Chromatin Association of Replicative α and ε DNA Polymerases in Fission Yeast

The tetrameric GINS complex, consisting of Sld5-Psf1-Psf2-Psf3, plays an essential role in the initiation and elongation steps of eukaryotic DNA replication, although its biochemical function is unclear. Here we investigate the function of GINS in fission yeast, using fusion of Psf1 and Psf2 subunits to a steroid hormone-binding domain (HBD) to make GINS function conditional on the presence of beta-estradiol. We show that inactivation of Psf1-HBD causes a tight but rapidly reversible DNA replication arrest phenotype. Inactivation of Psf2-HBD similarly blocks premeiotic DNA replication and leads to loss of nuclear localization of another GINS subunit, Psf3. Inactivation of GINS has distinct effects on the replication origin association and chromatin binding of two of the replicative DNA polymerases. Inactivation of Psf1 leads to loss of chromatin binding of DNA polymerase epsilon, and Cdc45 is similarly affected. In contrast, chromatin association of the catalytic subunit of DNA polymerase alpha is not affected by defective GINS function. We suggest that GINS functions in a pathway that involves Cdc45 and is necessary for DNA polymerase epsilon chromatin binding, but that a separate pathway sets up the chromatin association of DNA polymerase alpha.

The Drosophila Cdc6/18 protein has functions in both early and late S phase in S2 cells

The Cdc6/18 protein has been mainly characterised for its role in the initiation of DNA replication. Several studies exist, however, which suggest that it may also have a role in controlling the G2/M transition. Here we present studies on the Drosophila Cdc6 (DmCdc6) protein that support this dual function for the protein. First we show that its location is consistent with a cellular role post replication initiation as it remains nuclear throughout G1, S and G2 phases. In addition, we have been able to reduce the level of DmCdc6 protein to nondetectable levels in S2 cells using RNAi. This causes DNA fragmentation and cell cycle abnormalities which have some similarities with phenotypes previously observed in yeasts and are consistent with the cells entering mitosis with incompletely replicated DNA. Finally, we have stably overexpressed the DmCdc6 protein to a high level in S2 cells. Despite a large excess of protein the effects on the S2 cells were minimal. We did, however, detect a slight stalling of the cells in the late S phase of the cell cycle, which further supports the proposal that DmCdc6 has a role in controlling the transition from the S to M phases of the cycle.

The Human TPR Protein TTC4 Is a Putative Hsp90 Co-Chaperone Which Interacts with CDC6 and Shows Alterations in Transformed Cells

Background The human TTC4 protein is a TPR (tetratricopeptide repeat) motif-containing protein. The gene was originally identified as being localized in a genomic region linked to breast cancer and subsequent studies on melanoma cell lines revealed point mutations in the TTC4 protein that may be associated with the progression of malignant melanoma. Methodology/Principle Findings Here we show that TTC4 is a nucleoplasmic protein which interacts with HSP90 and HSP70, and also with the replication protein CDC6. It has significant structural and functional similarities with a previously characterised Drosophila protein Dpit47. We show that TTC4 protein levels are raised in malignant melanoma cell lines compared to melanocytes. We also see increased TTC4 expression in a variety of tumour lines derived from other tissues. In addition we show that TTC4 proteins bearing some of the mutations previously identified from patient samples lose their interaction with the CDC6 protein. Conclusions/Significance Based on these results and our previous work with the Drosophila Dpit47 protein we suggest that TTC4 is an HSP90 co-chaperone protein which forms a link between HSP90 chaperone activity and DNA replication. We further suggest that the loss of the interaction with CDC6 or with additional client proteins could provide one route through which TTC4 could influence malignant development of cells.

DNAReplication: a database of information and resources for the eukaryotic DNA replication community

DNAReplication (at http://www.dnareplication.net) has been set up as a freely available single resource to facilitate access to information on eukaryotic DNA replication. This database summarizes organism-sorted data on replication proteins in the categories of nomenclature, biochemical properties, motifs, interactions, modifications, structure, cell localization and expression, and general comments. Replication concepts are defined and a general model of the steps in DNA replication is presented. Links to relevant websites and homepages of replication labs are provided. The site also has an interactive section where links to recent replication papers are posted and readers are provided with the facility to post comments about each paper. The interactive and links pages are modified weekly and the whole site is updated annually.

Identification and characterisation of the Drosophila homologue of the yeast Uba2 gene.

We have identified the Drosophila uba2 protein (dUba2). Analysis of the amino acid composition reveals similarity with both the mammalian (47% identity) and yeast (31% identity) homologues. dUba2 is present throughout the Drosophila life cycle but is most abundant during stages of proliferation. The protein is nucleoplasmic throughout much of the cell cycle, however it is lost from the nucleus during mitosis. The DUba2 localisation in the nucleoplasm is not uniform but is observed as concentrated patches reminiscent of the staining patterns seen for other proteins from this group. The nature of these sites is not clear, however the failure of dUba2 to localise to the sites of chorion amplification in ovaries suggests that they are not sites of ongoing DNA replication.

Forced binding of the origin of replication complex to chromosomal sites in Drosophila S2 cells creates an origin of replication

Origins of replication in higher eukaryotes appear to lack specific sequence characteristics and those mapped often appear to be spread over several kilobases. This has complicated the study of site-specific events at origins of replication in vivo. Here we show that fusion of a Gal4-binding domain to proteins of the origin of replication complex (Orc) is sufficient to direct initiation to Gal4-binding sites inserted in the Drosophila S2 cell chromosome. The activation appears to go via an authentic route, taking place only in the S phase of the cell cycle and involving the formation of a prereplication complex. We have also shown that the origin-associated acetylation of histone H4 at K12 can be directed to the region of Orc binding by the presence of Orc. We expect that this system can provide a useful tool for the study of site-specific events at origins of replication in higher eukaryotes and a means to dissect Orc-dependent and Orc-independent events at origins.