Telomere and ribosomal DNA repeats are chromosomal targets of the bloom syndrome DNA helicase - PubMed (original) (raw)
Telomere and ribosomal DNA repeats are chromosomal targets of the bloom syndrome DNA helicase
James Schawalder et al. BMC Cell Biol. 2003.
Abstract
Background: Bloom syndrome is one of the most cancer-predisposing disorders and is characterized by genomic instability and a high frequency of sister chromatid exchange. The disorder is caused by loss of function of a 3' to 5' RecQ DNA helicase, BLM. The exact role of BLM in maintaining genomic integrity is not known but the helicase has been found to associate with several DNA repair complexes and some DNA replication foci.
Results: Chromatin immunoprecipitation of BLM complexes recovered telomere and ribosomal DNA repeats. The N-terminus of BLM, required for NB localization, is the same as the telomere association domain of BLM. The C-terminus is required for ribosomal DNA localization. BLM localizes primarily to the non-transcribed spacer region of the ribosomal DNA repeat where replication forks initiate. Bloom syndrome cells expressing the deletion alleles lacking the ribosomal DNA and telomere association domains have altered cell cycle populations with increased S or G2/M cells relative to normal.
Conclusion: These results identify telomere and ribosomal DNA repeated sequence elements as chromosomal targets for the BLM DNA helicase during the S/G2 phase of the cell cycle. BLM is localized in nuclear bodies when it associates with telomeric repeats in both telomerase positive and negative cells. The BLM DNA helicase participates in genomic stability at ribosomal DNA repeats and telomeres.
Figures
Figure 1
Analysis of rDNA and telomere repeats in normal and BS lymphoblastoid cell lines. A. Diagram of experimental design [28]. B. Relative amounts of total rDNA and telomere DNA in the clones from the normal (HG1522) and BS (HG1525) cell lines using a Perkin-Elmer (ABI) kit for rDNA or rRNA and membrane-transfer hybridization of total genomic DNA for telomere DNA.
Figure 2
BLM mutation map and telomere association domains. A. Map of BLM mutations [19] and domains with assigned sequence identities. NLS indicates the basic nuclear localization signal [60]. The HRD (
H
elicase and
R
NAse
D
) region is common to several members of the RecQ DNA helicase family and structural modeling suggests it is a single-strand nucleic acid binding domain [31]. The red bars indicate regions with a high density of negatively charged amino acids [3]. B. ChIP mapping of the telomere association domains of BLM. Equivalent amounts of DNA recovered from the induced cell lines were denatured and transferred to a nylon membrane. Telomere repeats sequences were quantitated by hybridization with a radiolabeled complementary oligonucleotide [10] and exposure to a phosphorimager screen. Hybridization units were calculated relative to the cell line expressing EGFP alone. Values are the average of two experiments: BLM 8+/-1.4, ΔN1 7+/-0.1, ΔN2 4+/-2.8, ΔN3 1+/-0.1, ΔN4 2+/-1.4, K695T 5+/-2.8, ΔH1 5+/-0.1, ΔC1 6+/-1.4, ΔC2 7+/-1.4.
Figure 3
Map of sequence elements in the human rDNA repeat unit and identification of the BLM association domains. A. The human rDNA repeat unit (U13369) sequence features and the location of the region isolated with a polyclonal BLM antibody. The orange arrows indicate sites of primer sets. The dotted lines indicate the direction of DNA replication origins [30]. B. ChIP mapping of BLM association with the rDNA repeat and mapping of BLM domains required for rDNA association. Equivalent amounts of DNA recovered from the induced cell lines were amplified with Taq DNA polymerase and one of eight primer sets, denatured and transferred to a nylon membrane. The PCR products were hybridized with the radiolabeled forward primer in each set and quantitated using a phosphorimager. Hybridization units were calculated relative to the cell line expressing EGF alone. C. A simple model for the BLM-binding region of the rDNA repeat.
Figure 4
Expression and purification of GFP-BLM alleles in Sf9 cells. A. Fluorescent micrographs of Sf9 cells infected with recombinant baculoviruses expressing GFP-BLM deletion proteins constructed using methods described previously for the full length normal BLM [19]. The ΔN3 image contains a bi-nucleated cell. B. Silver-stained gels of GFP-BLM deletion proteins isolated in the same manner as normal GFP-BLM [19]. Similar volumes of peak fractions are shown to demonstrate the relative recoveries of the different alleles.
Figure 5
Helicase activity of the GFP-BLM deletion proteins. A. Autoradiogram of the unwinding assay gel. The 0 lane contains no enzyme. The Δlane reaction was heated at 95°C for 5 minutes. The C2 reaction contains 1 ng protein and other reactions with the deletion proteins contain 25 ng. The normal BLM reactions contain the amounts shown in the right graph in panel B. Activities are reported relative to 8 units/ng DNA-dependent ATPase activity and 7 units/ng unwinding activity for normal GFP-BLM. B. Graphic representation of helicase activities relative to normal GFP-BLM. Assays were performed as described previously [4]. Quantitation of the autoradiogram using a phosphorimager is shown in the left and right graphs. Relative DNA-dependent ATPase activities are shown in the left graph. The thin layer ATPase assays are not shown.
Figure 6
Cell cycle assay of induced cell lines. Cell lines expressing GFP-BLM alleles described previously [19] were induced for five days with 1 μg/ml doxycycline. Cell cultures were harvested, fixed and stained with PI. Relative DNA content was measured by FACS analysis. A. Comparison of DNA content profiles of the cell lines expressing the normal and N-terminal deletion alleles. B. Comparison of DNA content profiles of the cell lines expressing the normal, helicase and C-terminal mutant alleles.
Figure 7
Quadriradial formation and gene density. A. Data from a study of 481 quadriradials found in BS lymphocytes [34] is shown as the number of quadriradials observed relative to the length of each chromosome. These values are compared to gene density on each of the 22 human autosomal chromosomes as calculated from the public human genome database [27]. The number of each chromosome is shown above the gene density bar. B-D. Model of origin of quadriradial composed of two acrocentric chromosomes that have replicated in the presence of BrdU and stained for SCE analysis. The rDNA repeats are in close physical proximity, possibly in the nucleolus, and have numerous single-stranded invasions that are not resolved. During DNA replication the sister chromatids can develop entanglements as well. These unresolved invasions combined with the attachments between the sister chromatids lead to mitotic chiasmata. Quadriradial image from Schonberg [61].
References
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