Most eukaryotic organisms employ a telomerase complex for the maintenance of chromosome ends. The core of this complex is composed of telomerase reverse transcriptase (TERT) and telomerase RNA (TR) subunits. The TERT reverse transcriptase (RT) domain synthesises telomeric DNA using the TR template sequence. The other TERT domains contribute to this process in different ways. In particular, the TERT RNA-binding domain (TRBD) interacts with specific TR motif(s). Using a yeast 3-hybrid system, we show the critical role of Arabidopsis thaliana (At) TRBD and embryophyta-conserved KRxR motif in the unstructured linker preceding the TRBD domain for binding to the recently identified AtTR subunit. We also show the essential role of the predicted P4 stem and pseudoknot AtTR structures and provide evidence for the binding of AtTRBD to pseudoknot and KRxR motif stabilising interaction with the P4 stem structure. Our results thus provide the first insight into the core part of the plant telomerase complex.

• Keywords
• A.thaliana telomerase, AtTERT, AtTR, Protein-RNA interactions, Yeast three-hybrid,
• MeSH
• Arabidopsis * genetics   enzymology   MeSH
• Nucleic Acid Conformation MeSH
• Arabidopsis Proteins * genetics   metabolism   chemistry   MeSH
• RNA, Plant genetics   metabolism   MeSH
• RNA metabolism   genetics   MeSH
• Two-Hybrid System Techniques MeSH
• Telomerase * genetics   metabolism   chemistry   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH

BACKGROUND: Structural Maintenance of Chromosomes (SMC) complexes are molecular machines driving chromatin organization at higher levels. In eukaryotes, three SMC complexes (cohesin, condensin and SMC5/6) play key roles in cohesion, condensation, replication, transcription and DNA repair. Their physical binding to DNA requires accessible chromatin. RESULTS: We performed a genetic screen in fission yeast to identify novel factors required for SMC5/6 binding to DNA. We identified 79 genes of which histone acetyltransferases (HATs) were the most represented. Genetic and phenotypic analyses suggested a particularly strong functional relationship between the SMC5/6 and SAGA complexes. Furthermore, several SMC5/6 subunits physically interacted with SAGA HAT module components Gcn5 and Ada2. As Gcn5-dependent acetylation facilitates the accessibility of chromatin to DNA-repair proteins, we first analysed the formation of DNA-damage-induced SMC5/6 foci in the Δgcn5 mutant. The SMC5/6 foci formed normally in Δgcn5, suggesting SAGA-independent SMC5/6 localization to DNA-damaged sites. Next, we used Nse4-FLAG chromatin-immunoprecipitation (ChIP-seq) analysis in unchallenged cells to assess SMC5/6 distribution. A significant portion of SMC5/6 accumulated within gene regions in wild-type cells, which was reduced in Δgcn5 and Δada2 mutants. The drop in SMC5/6 levels was also observed in gcn5-E191Q acetyltransferase-dead mutant. CONCLUSION: Our data show genetic and physical interactions between SMC5/6 and SAGA complexes. The ChIP-seq analysis suggests that SAGA HAT module targets SMC5/6 to specific gene regions and facilitates their accessibility for SMC5/6 loading.

• Keywords
• Ada2, Chromatin accessibility, DNA repair, Gcn5, Gene regions, Genetic and protein–protein interactions, Histone H3K9ac acetylation, Nse3 KITE, SAGA histone acetyltransferase module, SMC5/6 complex targeting, rDNA,
• MeSH
• Acetyltransferases genetics   MeSH
• Cell Nucleus metabolism   MeSH
• Chromatin metabolism   MeSH
• Chromosomes metabolism   MeSH
• DNA metabolism   MeSH
• Histone Acetyltransferases genetics   metabolism   MeSH
• Cell Cycle Proteins genetics   metabolism   MeSH
• Schizosaccharomyces pombe Proteins * genetics   metabolism   MeSH
• Schizosaccharomyces * genetics   metabolism   MeSH
• Carrier Proteins metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Acetyltransferases MeSH
• Chromatin MeSH
• DNA MeSH
• Gcn5 protein, S pombe MeSH Browser
• Histone Acetyltransferases MeSH
• Nse4 protein, S pombe MeSH Browser
• Cell Cycle Proteins MeSH
• Schizosaccharomyces pombe Proteins * MeSH
• Smc5 protein, S pombe MeSH Browser
• smc6 protein, S pombe MeSH Browser
• Carrier Proteins MeSH

The SMC (Structural Maintenance of Chromosomes) complexes are composed of SMC dimers, kleisin and kleisin-interacting (HAWK or KITE) subunits. Mutual interactions of these subunits constitute the basal architecture of the SMC complexes. In addition, binding of ATP molecules to the SMC subunits and their hydrolysis drive dynamics of these complexes. Here, we developed new systems to follow the interactions between SMC5/6 subunits and the relative stability of the complex. First, we show that the N-terminal domain of the Nse4 kleisin molecule binds to the SMC6 neck and bridges it to the SMC5 head. Second, binding of the Nse1 and Nse3 KITE proteins to the Nse4 linker increased stability of the ATP-free SMC5/6 complex. In contrast, binding of ATP to SMC5/6 containing KITE subunits significantly decreased its stability. Elongation of the Nse4 linker partially suppressed instability of the ATP-bound complex, suggesting that the binding of the KITE proteins to the Nse4 linker constrains its limited size. Our data suggest that the KITE proteins may shape the Nse4 linker to fit the ATP-free complex optimally and to facilitate opening of the complex upon ATP binding. This mechanism suggests an important role of the KITE subunits in the dynamics of the SMC5/6 complexes.

• MeSH
• Adenosine Triphosphatases metabolism   MeSH
• Nuclear Proteins genetics   metabolism   MeSH
• Macromolecular Substances metabolism   MeSH
• Mutagenesis, Site-Directed MeSH
• Cell Cycle Proteins genetics   metabolism   MeSH
• Schizosaccharomyces pombe Proteins genetics   metabolism   MeSH
• Schizosaccharomyces genetics   metabolism   MeSH
• Sequence Alignment MeSH
• Two-Hybrid System Techniques MeSH
• Carrier Proteins genetics   metabolism   MeSH
• Protein Binding genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine Triphosphatases MeSH
• Nuclear Proteins MeSH
• Macromolecular Substances MeSH
• Nse1 protein, S pombe MeSH Browser
• Nse3 protein, S pombe MeSH Browser
• Nse4 protein, S pombe MeSH Browser
• Cell Cycle Proteins MeSH
• Schizosaccharomyces pombe Proteins MeSH
• Smc5 protein, S pombe MeSH Browser
• smc6 protein, S pombe MeSH Browser
• Carrier Proteins MeSH