Human RecQ5beta, a large isomer of RecQ5 DNA helicase, localizes in the nucleoplasm and interacts with topoisomerases 3alpha and 3beta - PubMed (original) (raw)
Comparative Study
Human RecQ5beta, a large isomer of RecQ5 DNA helicase, localizes in the nucleoplasm and interacts with topoisomerases 3alpha and 3beta
A Shimamoto et al. Nucleic Acids Res. 2000.
Abstract
The RecQ helicase superfamily has been implicated in DNA repair and recombination. At least five human RecQ-related genes exist: RecQ1, BLM, WRN, RecQ4 and RecQ5. Mutations in BLM, WRN and RecQ4 are associated with Bloom, Werner and Rothmund-Thomson syndromes, respectively, involving a predisposition to malignancies and a cellular phenotype that includes increased chromosome instability. RecQ5 is small, containing only a core part of the RecQ helicase, but three isomer transcripts code for small RecQ5alpha (corresponding to the original RecQ5 with 410 amino acids), new large RecQ5beta (991 amino acids) and small RecQ5gamma (435 amino acids) proteins that contain the core helicase motifs. By determining the genomic structure, we found that the three isoforms are generated by differential splicing from the RecQ5 gene that contains at least 19 exons. Northern blot analysis using a RecQ5beta-specific probe indicates that RecQ5beta mRNA is expressed strongly in the testis. Immunocytochemical staining of three N-terminally tagged RecQ5 isomers expressed in 293EBNA cells showed that RecQ5beta migrates to the nucleus and exists exclusively in the nucleoplasm, while the small RecQ5alpha and RecQ5gamma proteins stay in the cytoplasm. Immunoprecipitation and an extended cytochemical experiment suggested that the nucleoplasmic RecQ5beta, like yeast Sgs1 DNA helicase, binds to topoisomerases 3alpha and 3beta, but not to topoisomerase 1. These results predict that RecQ5beta may have an important role in DNA metabolism and may also be related to a distinct genetic disease.
Figures
Figure 1
Structures of RecQ5 genes and the encoded RecQ5 proteins. (a) Genomic structure of the RecQ5 gene and the exon uses of three RecQ5 mRNA isomers, RecQ5α, RecQ5β and RecQ5γ. (b) Schematic representation of three RecQ5 helicase isomers, RecQ5α, RecQ5β and RecQ5γ. The sizes of each protein are given on the right. The darkened areas indicate the locations of the helicase domains shared by the RecQ helicase family. The hatched areas indicate the extended homologous region to E.coli RecQ helicase. The regions indicated by bars indicate identical amino acid sequences among the three isoforms.
Figure 2
Comparison of the amino acid sequences of Drosophila melanogaster RecQ5a and human RecQ5β. dRecQ5a, Drosophila melanogaster RecQ5a; hRecQ5β, human RecQ5β. The asterisks denote amino acid residues identical between the two proteins.
Figure 3
Multiple tissue northern blot analysis to compare the expression levels of the human RecQ5β gene. A multiple tissue northern blot (Clontech) was prepared with 2 µg/lane poly(A)+ RNA. A DNA fragment containing exons 14–19 of the RecQ5β gene was used for the probe. Arrows show the major species of RecQ5β transcripts of 3.6 and 3.8 kb that were detected in this study and in our previous report (10). Asterisks show the RecQ5β 3′-terminus-related RNA species that were not pursued in this study.
Figure 4
Expression of RecQ5 isoforms in 293EBNA cells. (a) Schematic representations of three recombinant RecQ5 helicase isomers, RecQ5α, RecQ5β and RecQ5γ, all N-terminally tagged with influenza virus 6xHA. RecQ5βc contains an N-terminal 6xHA tag and the C-terminal region of RecQ5β (amino acid residues 746–991). Numbers indicate amino acid residues. (b) Western blot analysis of three RecQ5 helicase isomers and RecQ5βc expressed in 293EBNA cells. Cell lysates prepared from cells transiently transfected with each construct were size fractionated by SDS–PAGE and analyzed by western blot analysis using anti-HA antibody.
Figure 5
Subcellular localization of RecQ5 isoforms in 293EBNA cells. Each construct was transiently expressed in 293EBNA cells and the cells fixed and stained with anti-HA antibody as shown in Materials and Methods. Localization of the RecQ5 isoform and RecQ5βc proteins are indicated by FITC (green, left panel) and the nuclear positions are shown by DAPI (blue, right panel). (a and b) RecQ5α; (c and d) RecQ5β; (e and f) RecQ5γ; (g and h) RecQ5βc.
Figure 6
Co-immunoprecipitation of RecQ5β and human topoisomerases 3α and 3β. (a) Expression of 6xHA-tagged RecQ5β protein in 293EBNA cells. 293EBNA cells were transfected with 6xHA–RecQ5β, Flag–Top3α and Flag–Top3β as indicated. Cell lysates were prepared from the cells, size fractionated by SDS–PAGE and analyzed by western blot analysis using anti-HA antibodies. (b) Expression of Flag-tagged type I topoisomerase proteins in 293EBNA cells. Anti-Flag antibodies were used in a western blot analysis. (c) RecQ5β protein co-immunoprecipitated with topoisomerases 3α and 3β. Type I topoisomerases and their binding proteins were precipitated with anti-Flag IgG-conjugated agarose beads and were eluted with Flag peptide. The eluted proteins were size fractionated by SDS–PAGE and analyzed by western blot analysis using anti-HA antibodies.
Figure 7
Co-localization of RecQ5β with topoisomerases 3α and 3β. 293EBNA cells were co-transfected with 6×HA–RecQ5β and Flag–Top3α (a) or Flag–Top3β (b), then fixed and stained with anti-HA and anti-Flag antibodies as described in Materials and Methods. Flag–Top3α and Flag–Top3β proteins were detected by FITC (green, left panels) and 6×HA–RecQ5β protein by Texas red (red, center panels). The merged profiles are shown in the right panels. Co-localization of RecQ5β with topoisomerases 3α and 3β is seen in yellow in the right panels.
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