Reversal of terminal differentiation and control of DNA replication

DNA replication in mammalian cells occurs in discrete nuclear foci. Here we show that terminally differentiated myotubes can be induced to reenter S phase and show the same pattern of replication foci as cycling cells. We used this cellular system to analyze the interaction of cell cycle proteins with these foci in vivo. Cyclin A and cdk2, but not cyclin B1 and cdc2, were specifically localized at nuclear replication foci, just like the replication protein proliferating cell nuclear antigen. A potential target of cyclin A and cdk2 is the 34 kd subunit of replication protein A (RPA34). In contrast with the 70 kd subunit, which localizes to the foci, RPA34 was not detected at these replication sites, which may reflect a transient interaction. The specific localization of cyclin A and cdk2 at nuclear replication foci provides a direct link between cell cycle regulation and DNA replication

XRCCI and PCNA are loading platforms with distinct kinetic properties and different capacities to respond to multiple DNA lesions

Background: Genome integrity is constantly challenged and requires the coordinated recruitment of multiple enzyme activities to ensure efficient repair of DNA lesions. We investigated the dynamics of XRCC1 and PCNA that act as molecular loading platforms and play a central role in this coordination. Results: Local DNA damage was introduced by laser microirradation and the recruitment of fluorescent XRCC1 and PCNA fusion proteins was monitored by live cell microscopy. We found an immediate and fast recruitment of XRCC1 preceding the slow and continuous recruitment of PCNA. Fluorescence bleaching experiments ( FRAP and FLIP) revealed a stable association of PCNA with DNA repair sites, contrasting the high turnover of XRCC1. When cells were repeatedly challenged with multiple DNA lesions we observed a gradual depletion of the nuclear pool of PCNA, while XRCC1 dynamically redistributed even to lesions inflicted last. Conclusion: These results show that PCNA and XRCC1 have distinct kinetic properties with functional consequences for their capacity to respond to successive DNA damage events

Intrinsic and Extrinsic Connections of Tet3 Dioxygenase with CXXC Zinc Finger Modules.

Tet proteins are emerging as major epigenetic modulators of cell fate and plasticity. However, little is known about how Tet proteins are targeted to selected genomic loci in distinct biological contexts. Previously, a CXXC-type zinc finger domain in Tet1 was shown to bind CpG-rich DNA sequences. Interestingly, in human and mouse the Tet2 and Tet3 genes are adjacent to Cxxc4 and Cxxc10-1, respectively. The CXXC domains encoded by these loci, together with those in Tet1 and Cxxc5, identify a distinct homology group within the CXXC domain family. Here we provide evidence for alternative mouse Tet3 transcripts including the Cxxc10-1 sequence (Tet3(CXXC)) and for an interaction between Tet3 and Cxxc4. In vitro Cxxc4 and the isolated CXXC domains of Tet1 and Tet3(CXXC) bind DNA substrates with similar preference towards the modification state of cytosine at a single CpG site. In vivo Tet1 and Tet3 isoforms with and without CXXC domain hydroxylate genomic 5-methylcytosine with similar activity. Relative transcript levels suggest that distinct ratios of Tet3(CXXC) isoforms and Tet3-Cxxc4 complex may be present in adult tissues. Our data suggest that variable association with CXXC modules may contribute to context specific functions of Tet proteins

Interactions within the mammalian DNA methyltransferase family

BACKGROUND: In mammals, epigenetic information is established and maintained via the postreplicative methylation of cytosine residues by the DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b. Dnmt1 is required for maintenance methylation whereas Dnmt3a and Dnmt3b are responsible for de novo methylation. Contrary to Dnmt3a or Dnmt3b, the isolated C-terminal region of Dnmt1 is catalytically inactive, despite the presence of the sequence motifs typical of active DNA methyltransferases. Deletion analysis has revealed that a large part of the N-terminal domain is required for enzymatic activity. RESULTS: The role played by the N-terminal domain in this regulation has been investigated using the yeast two-hybrid system. We show here the presence of an intra-molecular interaction in Dnmt1 but not in Dnmt3a or Dnmt3b. This interaction was confirmed by immunoprecipitation and was localized by deletion mapping. Furthermore, a systematic analysis of interactions among the Dnmt family members has revealed that DNMT3L interacts with the C-terminal domain of Dnmt3a and Dnmt3b. CONCLUSIONS: The lack of methylating ability of the isolated C-terminal domain of Dnmt1 could be explained in part by a physical interaction between N- and C-terminal domains that apparently is required for activation of the catalytic domain. Our deletion analysis suggests that the tertiary structure of Dnmt1 is important in this process rather than a particular sequence motif. Furthermore, the interaction between DNMT3L and the C-terminal domains of Dnmt3a and Dnmt3b suggests a mechanism whereby the enzymatically inactive DNMT3L brings about the methylation of its substrate by recruiting an active methylase

Differential recruitment of DNA Ligase I and III to DNA repair sites

DNA ligation is an essential step in DNA replication, repair and recombination. Mammalian cells contain three DNA Ligases that are not interchangeable although they use the same catalytic reaction mechanism. To compare the recruitment of the three eukaryotic DNA Ligases to repair sites in vivo we introduced DNA lesions in human cells by laser microirradiation. Time lapse microscopy of fluorescently tagged proteins showed that DNA Ligase III accumulated at microirradiated sites before DNA Ligase I, whereas we could detect only a faint accumulation of DNA Ligase IV. Recruitment of DNA Ligase I and III to repair sites was cell cycle independent. Mutational analysis and binding studies revealed that DNA Ligase I was recruited to DNA repair sites by interaction with PCNA while DNA Ligase III was recruited via its BRCT domain mediated interaction with XRCC1. Selective recruitment of specialized DNA Ligases may have evolved to accommodate the particular requirements of different repair pathways and may thus enhance efficiency of DNA repair

PCNA acts as a stationary loading platform for transiently interacting Okazaki fragment maturation proteins

In DNA replication, the leading strand is synthesized continuously, but lagging strand synthesis requires the complex, discontinuous synthesis of Okazaki fragments, and their subsequent joining. We have used a combination of in situ extraction and dual color photobleaching to compare the dynamic properties of three proteins essential for lagging strand synthesis: the polymerase clamp proliferating cell nuclear antigen (PCNA) and two proteins that bind to it, DNA Ligase I and Fen1. All three proteins are localized at replication foci (RF), but in contrast to PCNA, Ligase and Fen1 were readily extracted. Dual photobleaching combined with time overlays revealed a rapid exchange of Ligase and Fen1 at RF, which is consistent with de novo loading at every Okazaki fragment, while the slow recovery of PCNA mostly occurred at adjacent, newly assembled RF. These data indicate that PCNA works as a stationary loading platform that is reused for multiple Okazaki fragments, while PCNA binding proteins only transiently associate and are not stable components of the replication machinery

Direct and dynamic detection of HIV-1 in living cells.

In basic and applied HIV research, reliable detection of viral components is crucial to monitor progression of infection. While it is routine to detect structural viral proteins in vitro for diagnostic purposes, it previously remained impossible to directly and dynamically visualize HIV in living cells without genetic modification of the virus. Here, we describe a novel fluorescent biosensor to dynamically trace HIV-1 morphogenesis in living cells. We generated a camelid single domain antibody that specifically binds the HIV-1 capsid protein (CA) at subnanomolar affinity and fused it to fluorescent proteins. The resulting fluorescent chromobody specifically recognizes the CA-harbouring HIV-1 Gag precursor protein in living cells and is applicable in various advanced light microscopy systems. Confocal live cell microscopy and super-resolution microscopy allowed detection and dynamic tracing of individual virion assemblies at the plasma membrane. The analysis of subcellular binding kinetics showed cytoplasmic antigen recognition and incorporation into virion assembly sites. Finally, we demonstrate the use of this new reporter in automated image analysis, providing a robust tool for cell-based HIV research

The carcinogenic effect of various multi-walled carbon nanotubes (MWCNTs) after intraperitoneal injection in rats

Background: Biological effects of tailor-made multi-walled carbon nanotubes (MWCNTs) without functionalization were investigated in vivo in a two-year carcinogenicity study. In the past, intraperitoneal carcinogenicity studies in rats using biopersistent granular dusts had always been negative, whereas a number of such studies with different asbestos fibers had shown tumor induction. The aim of this study was to identify possible carcinogenic effects of MWCNTs. We compared induced tumors with asbestos-induced mesotheliomas and evaluated their relevance for humans by immunohistochemical methods. Methods: A total of 500 male Wistar rats (50 per group) were treated once by intraperitoneal injection with 109 or 5 � 109 WHO carbon nanotubes of one of four different MWCNTs suspended in artificial lung medium, which was also used as negative control. Amosite asbestos (108 WHO fibers) served as positive control. Morbid rats were sacrificed and necropsy comprising all organs was performed. Histopathological classification of tumors and, additionally, immunohistochemistry were conducted for podoplanin, pan-cytokeratin, and vimentin to compare induced tumors with malignant mesotheliomas occurring in humans. Results: Treatments induced tumors in all dose groups, but incidences and times to tumor differed between groups. Most tumors were histologically and immunohistochemically classified as malignant mesotheliomas, revealing a predominantly superficial spread on the serosal surface of the abdominal cavity. Furthermore, most tumors showed invasion of peritoneal organs, especially the diaphragm. All tested MWCNT types caused mesotheliomas. We observed highest frequencies and earliest appearances after treatment with the rather straight MWCNT types A and B. In the MWCNT C groups, first appearances of morbid mesothelioma-bearing rats were only slightly later. Later during the two-year study, we found mesotheliomas also in rats treated with MWCNT D - the most curved type of nanotubes. Malignant mesotheliomas induced by intraperitoneal injection of different MWCNTs and of asbestos were histopathologically and immunohistochemically similar, also compared with mesotheliomas in man, suggesting similar pathogenesis. Conclusion: We showed a carcinogenic effect for all tested MWCNTs. Besides aspect ratio, curvature seems to be an important parameter influencing the carcinogenicity of MWCNTs