Arabidopsis thaliana mutants in FAS1 and FAS2 subunits of chromatin assembly factor 1 (CAF1) show progressive loss of 45S rDNA copies and telomeres. We hypothesized that homology-dependent DNA damage repair (HDR) may contribute to the loss of these repeats in fas mutants. To test this, we generated double mutants by crossing fas mutants with knock-out mutants in RAD51B, one of the Rad51 paralogs of A. thaliana. Our results show that the absence of RAD51B decreases the rate of rDNA loss, confirming the implication of RAD51B-dependent recombination in rDNA loss in the CAF1 mutants. Interestingly, this effect is not observed for telomeric repeat loss, which thus differs from that acting in rDNA loss. Involvement of DNA damage repair in rDNA dynamics in fas mutants is further supported by accumulation of double-stranded breaks (measured as γ-H2AX foci) in 45S rDNA. Occurrence of the foci is not specific for S-phase, and is ATM-independent. While the foci in fas mutants occur both in the transcribed (intranucleolar) and non-transcribed (nucleoplasmic) fraction of rDNA, double fas rad51b mutants show a specific increase in the number of the intranucleolar foci. These results suggest that the repair of double-stranded breaks present in the transcribed rDNA region is RAD51B dependent and that this contributes to rDNA repeat loss in fas mutants, presumably via the single-stranded annealing recombination pathway. Our results also highlight the importance of proper chromatin assembly in the maintenance of genome stability.

• Keywords
• 45S rDNA, Arabidopsis thaliana, DNA repair, FAS1, FAS2, RAD51B, chromatin assembly factor 1, genome instability,
• MeSH
• Arabidopsis genetics   metabolism   MeSH
• Chromatin Assembly Factor-1 genetics   metabolism   MeSH
• Genomic Instability genetics   physiology   MeSH
• DNA Repair genetics   physiology   MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• DNA, Ribosomal genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chromatin Assembly Factor-1 MeSH
• FAS protein, Arabidopsis MeSH Browser
• Arabidopsis Proteins MeSH
• RAD51B protein, Arabidopsis MeSH Browser
• DNA, Ribosomal MeSH

Dysfunction of chromatin assembly factor 1 in FASCIATA mutants (fas) of Arabidopsis thaliana results in progressive loss of telomeric DNA. Although replicative telomere shortening is typically associated with incomplete resynthesis of their ends by telomerase, no change in telomerase activity could be detected in vitro in extracts from fas mutants. Besides a possible telomerase malfunction, the telomere shortening in fas mutants could presumably be due to problems with conventional replication of telomeres. To distinguish between the possible contribution of suboptimal function of telomerase in fas mutants under in vivo conditions and problems in conventional telomere replication, we crossed fas and tert (telomerase reverse transcriptase) knockout mutants and analyzed telomere shortening in segregated fas mutants, tert mutants, and double fas tert mutants in parallel. We demonstrate that fas tert knockouts show greater replicative telomere shortening than that observed even in the complete absence of telomerase (tert mutants). While the effect of tert and fas mutations on telomere lengths in double mutants is additive, manifestations of telomere dysfunction in double fas tert mutants (frequency of anaphase bridges, onset of chromosome end fusions, and common involvement of 45S rDNA in chromosome fusion sites) are similar to those in tert mutants. We conclude that in addition to possible impairment of telomerase action, a further mechanism contributes to telomere shortening in fas mutants.

• MeSH
• Arabidopsis enzymology   genetics   metabolism   MeSH
• Chromosomes, Plant genetics   metabolism   MeSH
• Chromatin Assembly Factor-1 genetics   metabolism   MeSH
• Mutation * MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• RNA Splicing Factors MeSH
• Telomerase genetics   metabolism   MeSH
• Telomere genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• At2g20020 protein, Arabidopsis MeSH Browser
• Chromatin Assembly Factor-1 MeSH
• FAS protein, Arabidopsis MeSH Browser
• Arabidopsis Proteins MeSH
• RNA Splicing Factors MeSH
• Telomerase MeSH
• TERT protein, Arabidopsis MeSH Browser

BACKGROUND: Telomeres, as elaborate nucleo-protein complexes, ensure chromosomal stability. When impaired, the ends of linear chromosomes can be recognised by cellular repair mechanisms as double-strand DNA breaks and can be healed by non-homologous-end-joining activities to produce dicentric chromosomes. During cell divisions, particularly during anaphase, dicentrics can break, thus producing naked chromosome tips susceptible to additional unwanted chromosome fusion. Many telomere-building protein complexes are associated with telomeres to ensure their proper capping function. It has been found however, that a number of repair complexes also contribute to telomere stability. RESULTS: We used Arabidopsis thaliana to study the possible functions of the DNA repair subunit, NBS1, in telomere homeostasis using knockout nbs1 mutants. The results showed that although NBS1-deficient plants were viable, lacked any sign of developmental aberration and produced fertile seeds through many generations upon self-fertilisation, plants also missing the functional telomerase (double mutants), rapidly, within three generations, displayed severe developmental defects. Cytogenetic inspection of cycling somatic cells revealed a very early onset of massive genome instability. Molecular methods used for examining the length of telomeres in double homozygous mutants detected much faster telomere shortening than in plants deficient in telomerase gene alone. CONCLUSIONS: Our findings suggest that NBS1 acts in concert with telomerase and plays a profound role in plant telomere renewal.

• MeSH
• Anaphase MeSH
• Arabidopsis cytology   enzymology   genetics   growth & development   MeSH
• Chromosomal Instability MeSH
• Chromosomes, Plant genetics   metabolism   MeSH
• Cytogenetic Analysis MeSH
• DNA-Binding Proteins genetics   metabolism   MeSH
• Telomere Homeostasis MeSH
• MRE11 Homologue Protein MeSH
• In Situ Hybridization, Fluorescence MeSH
• Nuclear Proteins genetics   metabolism   MeSH
• Germination MeSH
• Flowers cytology   genetics   metabolism   MeSH
• Protein Interaction Mapping MeSH
• Meiosis MeSH
• DNA Repair MeSH
• Cell Cycle Proteins genetics   metabolism   MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• Plant Cells enzymology   metabolism   MeSH
• Self-Fertilization MeSH
• Seeds genetics   growth & development   metabolism   MeSH
• Telomerase genetics   metabolism   MeSH
• Telomere genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA-Binding Proteins MeSH
• MRE11 Homologue Protein MeSH
• Nuclear Proteins MeSH
• Mre11 protein, Arabidopsis MeSH Browser
• Cell Cycle Proteins MeSH
• Arabidopsis Proteins MeSH
• rad50 protein, Arabidopsis MeSH Browser
• Telomerase MeSH
• TERT protein, Arabidopsis MeSH Browser

Chromatin Assembly Factor 1 (CAF1) is a three-subunit H3/H4 histone chaperone responsible for replication-dependent nucleosome assembly. It is composed of CAC 1-3 in yeast; p155, p60, and p48 in humans; and FASCIATA1 (FAS1), FAS2, and MULTICOPY SUPPRESSOR OF IRA1 in Arabidopsis thaliana. We report that disruption of CAF1 function by fas mutations in Arabidopsis results in telomere shortening and loss of 45S rDNA, while other repetitive sequences (5S rDNA, centromeric 180-bp repeat, CACTA, and Athila) are unaffected. Substantial telomere shortening occurs immediately after the loss of functional CAF1 and slows down at telomeres shortened to median lengths around 1 to 1.5 kb. The 45S rDNA loss is progressive, leaving 10 to 15% of the original number of repeats in the 5th generation of mutants affecting CAF1, but the level of the 45S rRNA transcripts is not altered in these mutants. Increasing severity of the fas phenotype is accompanied by accumulation of anaphase bridges, reduced viability, and plant sterility. Our results show that appropriate replication-dependent chromatin assembly is specifically required for stable maintenance of telomeres and 45S rDNA.

• MeSH
• Arabidopsis genetics   metabolism   MeSH
• DNA, Plant genetics   metabolism   MeSH
• Chromatin Assembly Factor-1 genetics   metabolism   MeSH
• Mutagenesis, Insertional MeSH
• Mutation MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• Gene Expression Regulation, Plant MeSH
• Chromatin Assembly and Disassembly MeSH
• DNA, Ribosomal genetics   metabolism   MeSH
• RNA, Ribosomal genetics   metabolism   MeSH
• Telomere metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Plant MeSH
• Chromatin Assembly Factor-1 MeSH
• FAS protein, Arabidopsis MeSH Browser
• Arabidopsis Proteins MeSH
• DNA, Ribosomal MeSH
• RNA, Ribosomal MeSH
• RNA, ribosomal, 45S MeSH Browser