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Characterization of BTBD1 and BTBD2, two similar BTB-domain-containing Kelch-like proteins that interact with Topoisomerase I.

Xu L, Yang L, Hashimoto K, Anderson M, Kohlhagen G, Pommier Y, D'Arpa P - BMC Genomics (2002)

Bottom Line: BTBD1 and BTBD2 are widely if not ubiquitously expressed in human tissues, and have two paralogs as well as putative orthologs in C. elegans and D. melanogaster.Epitope-tagged BTBD2 localized to cytoplasmic bodies.The characterization of BTBD1 and BTBD2 and their interaction with TOP1 is underway.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology Uniformed Services University of the Health Sciences 4301 Jones Bridge Road, Bethesda, MD 20814-4799, USA. lixu@usuhs.mil

ABSTRACT

Background: Two-hybrid screening for proteins that interact with the core domain of human topoisomerase I identified two novel proteins, BTBD1 and BTBD2, which share 80% amino acid identities.

Results: The interactions were confirmed by co-precipitation assays demonstrating the physical interaction of BTBD1 and BTBD2 with 100 kDa topoisomerase I from HeLa cells. Deletion mapping using two-hybrid and GST-pulldown assays demonstrated that less than the C-terminal half of BTBD1 is sufficient for binding topoisomerase I. The topoisomerase I sequences sufficient to bind BTBD2 were mapped to residues 215 to 329. BTBD2 with an epitope tag localized to cytoplasmic bodies. Using truncated versions that direct BTBD2 and TOP1 to the same cellular compartment, either the nucleus or the cytoplasm, co-localization was demonstrated in co-transfected Hela cells. The supercoil relaxation and DNA cleavage activities of topoisomerase I in vitro were affected little or none by co-incubation with BTBD2. Northern analysis revealed only a single sized mRNA for each BTBD1 and BTBD2 in all human tissues tested. Characterization of BTBD2 mRNA revealed a 255 nucleotide 90% GC-rich region predicted to encode the N-terminus. BTBD1 and BTBD2 are widely if not ubiquitously expressed in human tissues, and have two paralogs as well as putative orthologs in C. elegans and D. melanogaster.

Conclusions: BTBD1 and BTBD2 belong to a small family of uncharacterized proteins that appear to be specific to animals. Epitope-tagged BTBD2 localized to cytoplasmic bodies. The characterization of BTBD1 and BTBD2 and their interaction with TOP1 is underway.

No MeSH data available.


Localization of BTBD2 and co-localization with TOP1. HeLa cells were transfected with: A, GFP-tagged TOP1 lacking a nuclear localization (GFP-pTI-6); B, full length BTBD2 containing a C-terminal myc tag (m-BTBD2F); C, truncated BTBD2 having the N-terminal half the BTB domain deleted (m-BTBD2T); D, GFP-pTI-6 and m-BTBD2F; and E, GFP-pTI-6 and m-BTBD2T.
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Figure 6: Localization of BTBD2 and co-localization with TOP1. HeLa cells were transfected with: A, GFP-tagged TOP1 lacking a nuclear localization (GFP-pTI-6); B, full length BTBD2 containing a C-terminal myc tag (m-BTBD2F); C, truncated BTBD2 having the N-terminal half the BTB domain deleted (m-BTBD2T); D, GFP-pTI-6 and m-BTBD2F; and E, GFP-pTI-6 and m-BTBD2T.

Mentions: To determine the localization of BTBD2 in cells, we transiently transfected HeLa cells with expression plasmids encoding either myc-tagged full length BTBD2 (m-BTBD2F) or a truncated BTBD2 having the N-terminal half of the BTB domain removed (m-BTBD2T). The full length BTBD2 localized entirely to cytoplasmic bodies (Fig. 6B, m-BTBD2F). The truncated BTBD2 localized predominantly to nuclear bodies with a minor fraction localizing to cytoplasmic bodies (Fig. 6C, m-BTBD2T).


Characterization of BTBD1 and BTBD2, two similar BTB-domain-containing Kelch-like proteins that interact with Topoisomerase I.

Xu L, Yang L, Hashimoto K, Anderson M, Kohlhagen G, Pommier Y, D'Arpa P - BMC Genomics (2002)

Localization of BTBD2 and co-localization with TOP1. HeLa cells were transfected with: A, GFP-tagged TOP1 lacking a nuclear localization (GFP-pTI-6); B, full length BTBD2 containing a C-terminal myc tag (m-BTBD2F); C, truncated BTBD2 having the N-terminal half the BTB domain deleted (m-BTBD2T); D, GFP-pTI-6 and m-BTBD2F; and E, GFP-pTI-6 and m-BTBD2T.
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Related In: Results  -  Collection

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Figure 6: Localization of BTBD2 and co-localization with TOP1. HeLa cells were transfected with: A, GFP-tagged TOP1 lacking a nuclear localization (GFP-pTI-6); B, full length BTBD2 containing a C-terminal myc tag (m-BTBD2F); C, truncated BTBD2 having the N-terminal half the BTB domain deleted (m-BTBD2T); D, GFP-pTI-6 and m-BTBD2F; and E, GFP-pTI-6 and m-BTBD2T.
Mentions: To determine the localization of BTBD2 in cells, we transiently transfected HeLa cells with expression plasmids encoding either myc-tagged full length BTBD2 (m-BTBD2F) or a truncated BTBD2 having the N-terminal half of the BTB domain removed (m-BTBD2T). The full length BTBD2 localized entirely to cytoplasmic bodies (Fig. 6B, m-BTBD2F). The truncated BTBD2 localized predominantly to nuclear bodies with a minor fraction localizing to cytoplasmic bodies (Fig. 6C, m-BTBD2T).

Bottom Line: BTBD1 and BTBD2 are widely if not ubiquitously expressed in human tissues, and have two paralogs as well as putative orthologs in C. elegans and D. melanogaster.Epitope-tagged BTBD2 localized to cytoplasmic bodies.The characterization of BTBD1 and BTBD2 and their interaction with TOP1 is underway.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology Uniformed Services University of the Health Sciences 4301 Jones Bridge Road, Bethesda, MD 20814-4799, USA. lixu@usuhs.mil

ABSTRACT

Background: Two-hybrid screening for proteins that interact with the core domain of human topoisomerase I identified two novel proteins, BTBD1 and BTBD2, which share 80% amino acid identities.

Results: The interactions were confirmed by co-precipitation assays demonstrating the physical interaction of BTBD1 and BTBD2 with 100 kDa topoisomerase I from HeLa cells. Deletion mapping using two-hybrid and GST-pulldown assays demonstrated that less than the C-terminal half of BTBD1 is sufficient for binding topoisomerase I. The topoisomerase I sequences sufficient to bind BTBD2 were mapped to residues 215 to 329. BTBD2 with an epitope tag localized to cytoplasmic bodies. Using truncated versions that direct BTBD2 and TOP1 to the same cellular compartment, either the nucleus or the cytoplasm, co-localization was demonstrated in co-transfected Hela cells. The supercoil relaxation and DNA cleavage activities of topoisomerase I in vitro were affected little or none by co-incubation with BTBD2. Northern analysis revealed only a single sized mRNA for each BTBD1 and BTBD2 in all human tissues tested. Characterization of BTBD2 mRNA revealed a 255 nucleotide 90% GC-rich region predicted to encode the N-terminus. BTBD1 and BTBD2 are widely if not ubiquitously expressed in human tissues, and have two paralogs as well as putative orthologs in C. elegans and D. melanogaster.

Conclusions: BTBD1 and BTBD2 belong to a small family of uncharacterized proteins that appear to be specific to animals. Epitope-tagged BTBD2 localized to cytoplasmic bodies. The characterization of BTBD1 and BTBD2 and their interaction with TOP1 is underway.

No MeSH data available.