Molecular characterization of Chlamydomonas reinhardtii telomeres and telomerase mutants

International audienceTelomeres are repeated sequences found at the end of the linear chromosomes of most eukaryotes and are required for chromosome integrity. Expression of the reverse-transcriptase telo-merase allows for extension of telomeric repeats to counteract natural telomere shortening. Although Chlamydomonas rein-hardtii, a photosynthetic unicellular green alga, is widely used as a model organism in photosynthesis and flagella research, and for biotechnological applications, the biology of its telomeres has not been investigated in depth. Here, we show that the C. rein-hardtii (TTTTAGGG) n telomeric repeats are mostly nondegenerate and that the telomeres form a protective structure, with a subset ending with a 39 overhang and another subset presenting a blunt end. Although telomere size and length distributions are stable under various standard growth conditions, they vary substantially between 12 genetically close reference strains. Finally, we identify CrTERT, the gene encoding the catalytic subunit of telomerase and show that telomeres shorten progressively in mutants of this gene. Telomerase mutants eventually enter replicative senescence, demonstrating that telomerase is required for long-term maintenance of telomeres in C. reinhardtii

The SAP domain of Ku facilitates its efficient loading onto DNA ends

The evolutionarily conserved DNA repair complex Ku serves as the primary sensor of free DNA ends in eukaryotic cells. Its rapid association with DNA ends is crucial for several cellular processes, including non-homologous end joining (NHEJ) DNA repair and telomere protection. In this study, we conducted a transient kinetic analysis to investigate the impact of the SAP domain on individual phases of the Ku–DNA interaction. Specifically, we examined the initial binding, the subsequent docking of Ku onto DNA, and sliding of Ku along DNA. Our findings revealed that the C-terminal SAP domain of Ku70 facilitates the initial phases of the Ku–DNA interaction but does not affect the sliding process. This suggests that the SAP domain may either establish the first interactions with DNA, or stabilize these initial interactions during loading. To assess the biological role of the SAP domain, we generated Arabidopsis plants expressing Ku lacking the SAP domain. Intriguingly, despite the decreased efficiency of the ΔSAP Ku complex in loading onto DNA, the mutant plants exhibited full proficiency in classical NHEJ and telomere maintenance. This indicates that the speed with which Ku loads onto telomeres or DNA double-strand breaks is not the decisive factor in stabilizing these DNA structures.peer-reviewe

siRNA–Mediated Methylation of Arabidopsis Telomeres

Chromosome termini form a specialized type of heterochromatin that is important for chromosome stability. The recent discovery of telomeric RNA transcripts in yeast and vertebrates raised the question of whether RNA–based mechanisms are involved in the formation of telomeric heterochromatin. In this study, we performed detailed analysis of chromatin structure and RNA transcription at chromosome termini in Arabidopsis. Arabidopsis telomeres display features of intermediate heterochromatin that does not extensively spread to subtelomeric regions which encode transcriptionally active genes. We also found telomeric repeat–containing transcripts arising from telomeres and centromeric loci, a portion of which are processed into small interfering RNAs. These telomeric siRNAs contribute to the maintenance of telomeric chromatin through promoting methylation of asymmetric cytosines in telomeric (CCCTAAA)n repeats. The formation of telomeric siRNAs and methylation of telomeres relies on the RNA–dependent DNA methylation pathway. The loss of telomeric DNA methylation in rdr2 mutants is accompanied by only a modest effect on histone heterochromatic marks, indicating that maintenance of telomeric heterochromatin in Arabidopsis is reinforced by several independent mechanisms. In conclusion, this study provides evidence for an siRNA–directed mechanism of chromatin maintenance at telomeres in Arabidopsis

An investigation into computer-aided design of software

Manual software design methods suffer from many handicaps. As a result, the design documentation of software systems usually either does not exist, or is full of errors and out of date. Many software development, reliability, and maintainability problems reported in the literature can be traced to this state of affairs. The solution of these problems does not appear to lie in improved manual software design methods, but in the potentially much more "effective" computer-aided software design tools. This project set out to investigate how to aid the manual software design methods with a computer. A novel entity-relationship model for the software design specification was formulated and built into an interactive pilot software design tool. In spite of its simplicity, the model is capable of representing software structures from high-level architectural design to low-level detailed design. The tool stores the model entered by the user in a database. The model can then be inspected either on-line, or from printed documentation. The evaluation of the pilot tool, based on the feedback from the users, was favourable to the underlying model. However, many human-computer interface problems were identified. The effectiveness of software design tools was defined in terms of the designer productivity and the quality of the design documentation. An approach which consisted of repeated propositioning, implementation, and evaluation of modifications was then used to improve the effectiveness of the tool. A questionnaire was used to assess the opinion of the tool users more objectively, and an experiment was carried out to compare the effectiveness of the tool with a manual method. The results of this research have shown that the latest version of the tool is significantly more effective than the manual method

The biological functions of nonsense-mediated mRNA decay in plants

Nonsense-mediated mRNA decay (NMD) is an evolutionary conserved quality control pathway that inhibits expression of transcripts containing premature termination codon. Transcriptome and phenotypic studies across range of mutant organisms indicate roles of NMD beyond RNA quality control and imply its importance in regulating gene expression. Studies in Physcomitrella patens and Arabidopsis thaliana has shown that NMD is also important in plants where it contributes to regulation of pathogen defense, hormonal signaling, circadian clock, reproduction and gene evolution. Here we provide up to date overview of biological functions of NMD in plants. In addition, we discuss several biological processes where NMD factors implement their function through NMD-independent mechanisms

Characterization of DNA repair deficient strains of Chlamydomonas reinhardtii generated by insertional mutagenesis.

While the mechanisms governing DNA damage response and repair are fundamentally conserved, cross-kingdom comparisons indicate that they differ in many aspects due to differences in life-styles and developmental strategies. In photosynthetic organisms these differences have not been fully explored because gene-discovery approaches are mainly based on homology searches with known DDR/DNA repair proteins. Here we performed a forward genetic screen in the green algae Chlamydomonas reinhardtii to identify genes deficient in DDR/DNA repair. We isolated five insertional mutants that were sensitive to various genotoxic insults and two of them exhibited altered efficiency of transgene integration. To identify genomic regions disrupted in these mutants, we established a novel adaptor-ligation strategy for the efficient recovery of the insertion flanking sites. Four mutants harbored deletions that involved known DNA repair factors, DNA Pol zeta, DNA Pol theta, SAE2/COM1, and two neighbouring genes encoding ERCC1 and RAD17. Deletion in the last mutant spanned two Chlamydomonas-specific genes with unknown function, demonstrating the utility of this approach for discovering novel factors involved in genome maintenance