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Conserved overlapping gene arrangement, restricted expression, and biochemical activities of DNA polymerase ν (POLN).

Takata K, Tomida J, Reh S, Swanhart LM, Takata M, Hukriede NA, Wood RD - J. Biol. Chem. (2015)

Bottom Line: Consistent with this, injection of POLN-specific morpholino antisense oligonucleotides did not interfere with zebrafish embryonic development.These properties are conserved with the human enzyme.Although the physiological function of pol ν remains to be clarified, this study uncovers distinctive aspects of its expression control and evolutionarily conserved properties of this DNA polymerase.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Epigenetics and Molecular Carcinogenesis, University of Texas M.D. Anderson Cancer Center, Smithville, Texas 78957, the University of Texas Graduate School of Biomedical Sciences at Houston, Houston, Texas 77030, ktakata@mdanderson.org.

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A, POLN complex was immunopurified from nuclear extract prepared from HeLa S3 cells expressing FLAG-HA epitope-tagged POLN. Immunoblotting with specific antibodies confirmed the presence or absence of the proteins in the POLN complex. A single membrane was cut into sections as shown in supplemental Fig. S1 for immunoblotting with different antibodies. One of the membrane sections was first used for HELQ immunoblotting and then stripped using Thermo Restore Western Blot Stripping Buffer and reblotted for identification of FANCD2 and then again for POLN. B, POLN does not interact with the Fanconi core complex in the presence or absence of HU. The FANCL complex was immunopurified from nuclear extracts prepared from HeLa S3 cells expressing FLAG-HA epitope-tagged FANCL with or without 3 mm HU treatment for 24 h. The complex was sequentially purified with anti-FLAG and anti-HA antibodies. The complex was resolved by SDS-PAGE on a 4–20% gradient gel. Immunoblotting with specific antibodies confirmed the presence or absence of the proteins in the FANCL complex. FANCA but not POLN is present in the complex before and after HU treatment. Crude extract prepared from 293T cells transiently expressing POLN (lane labeled POLN) was used as a positive control for the POLN antibody.
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Figure 10: A, POLN complex was immunopurified from nuclear extract prepared from HeLa S3 cells expressing FLAG-HA epitope-tagged POLN. Immunoblotting with specific antibodies confirmed the presence or absence of the proteins in the POLN complex. A single membrane was cut into sections as shown in supplemental Fig. S1 for immunoblotting with different antibodies. One of the membrane sections was first used for HELQ immunoblotting and then stripped using Thermo Restore Western Blot Stripping Buffer and reblotted for identification of FANCD2 and then again for POLN. B, POLN does not interact with the Fanconi core complex in the presence or absence of HU. The FANCL complex was immunopurified from nuclear extracts prepared from HeLa S3 cells expressing FLAG-HA epitope-tagged FANCL with or without 3 mm HU treatment for 24 h. The complex was sequentially purified with anti-FLAG and anti-HA antibodies. The complex was resolved by SDS-PAGE on a 4–20% gradient gel. Immunoblotting with specific antibodies confirmed the presence or absence of the proteins in the FANCL complex. FANCA but not POLN is present in the complex before and after HU treatment. Crude extract prepared from 293T cells transiently expressing POLN (lane labeled POLN) was used as a positive control for the POLN antibody.

Mentions: It has been suggested that POLN interacts with several proteins, including HELQ, Fanconi anemia (FA) core complex proteins, and FANCD2 (16). It is intriguing that this set of proteins has testis-related functions. Helq knock-out male mice have significantly smaller testes (14), targeted disruption of several FA genes caused impaired fertility in mice (39), and infertility is a common feature among male FA patients (40). To test these proposed interactions and to uncover possible molecular pathways relevant to POLN, we searched for proteins with the potential to associate with POLN in vivo. POLN was stably expressed as a FLAG and HA epitope fusion (ePOLN) in HeLa S3 cells. ePOLN was recovered from nuclear extracts by sequential immunoprecipitation with anti-FLAG and anti-HA antibodies (28, 29). The immunoprecipitate was separated on a gradient gel (Fig. 9A), and proteins from gel sections were identified by liquid chromatography-mass spectrometry. The data were filtered to eliminate common s. Among the resulting top 150 ranking hits there were nine DNA repair-related proteins (supplemental Table 1). Six of these (BRCA1, BRCA2, BARD1, PALB2, FANCJ, and RBBP8/CtIP) are components of the A, B, and C BRCA1-associated complexes related to homologous recombination (Fig. 9A and Table 3) (41). None of these proteins were present in a control FLAG-HA purification from HeLa S3 cells transfected with empty control vector. In parallel experiments in the same system using FLAG-HA-tagged HELQ as bait, none of these proteins were detected in the HELQ complex (13). Although FA core complex proteins FANCD2 and HELQ were previously proposed as POLN-interacting partners (16), no peptides representing any of these proteins were identified as ePOLN-associated proteins (supplemental Table 1), just as POLN was not detected in the HELQ complex in the previous study (13). Immunoblotting confirmed that BRCA1 was present in the immunoprecipitate and that FANCD2 and HELQ were present in the input fraction but undetectable in the POLN immunoprecipitate (Fig. 10A). To confirm POLN-BRCA1 and POLN-FANCJ interactions in another cell line, V5-tagged POLN was coexpressed with FLAG-HA-tagged BRCA1 or FANCJ in 293T cells. After immunoprecipitation from whole-cell extract with anti-FLAG and anti-HA antibodies, V5 antibody was used to identify POLN in the immunoprecipitate. Co-immunoprecipitation was observed between POLN and BRCA1 (Fig. 9B) and POLN and FANCJ (Fig. 9C).


Conserved overlapping gene arrangement, restricted expression, and biochemical activities of DNA polymerase ν (POLN).

Takata K, Tomida J, Reh S, Swanhart LM, Takata M, Hukriede NA, Wood RD - J. Biol. Chem. (2015)

A, POLN complex was immunopurified from nuclear extract prepared from HeLa S3 cells expressing FLAG-HA epitope-tagged POLN. Immunoblotting with specific antibodies confirmed the presence or absence of the proteins in the POLN complex. A single membrane was cut into sections as shown in supplemental Fig. S1 for immunoblotting with different antibodies. One of the membrane sections was first used for HELQ immunoblotting and then stripped using Thermo Restore Western Blot Stripping Buffer and reblotted for identification of FANCD2 and then again for POLN. B, POLN does not interact with the Fanconi core complex in the presence or absence of HU. The FANCL complex was immunopurified from nuclear extracts prepared from HeLa S3 cells expressing FLAG-HA epitope-tagged FANCL with or without 3 mm HU treatment for 24 h. The complex was sequentially purified with anti-FLAG and anti-HA antibodies. The complex was resolved by SDS-PAGE on a 4–20% gradient gel. Immunoblotting with specific antibodies confirmed the presence or absence of the proteins in the FANCL complex. FANCA but not POLN is present in the complex before and after HU treatment. Crude extract prepared from 293T cells transiently expressing POLN (lane labeled POLN) was used as a positive control for the POLN antibody.
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Figure 10: A, POLN complex was immunopurified from nuclear extract prepared from HeLa S3 cells expressing FLAG-HA epitope-tagged POLN. Immunoblotting with specific antibodies confirmed the presence or absence of the proteins in the POLN complex. A single membrane was cut into sections as shown in supplemental Fig. S1 for immunoblotting with different antibodies. One of the membrane sections was first used for HELQ immunoblotting and then stripped using Thermo Restore Western Blot Stripping Buffer and reblotted for identification of FANCD2 and then again for POLN. B, POLN does not interact with the Fanconi core complex in the presence or absence of HU. The FANCL complex was immunopurified from nuclear extracts prepared from HeLa S3 cells expressing FLAG-HA epitope-tagged FANCL with or without 3 mm HU treatment for 24 h. The complex was sequentially purified with anti-FLAG and anti-HA antibodies. The complex was resolved by SDS-PAGE on a 4–20% gradient gel. Immunoblotting with specific antibodies confirmed the presence or absence of the proteins in the FANCL complex. FANCA but not POLN is present in the complex before and after HU treatment. Crude extract prepared from 293T cells transiently expressing POLN (lane labeled POLN) was used as a positive control for the POLN antibody.
Mentions: It has been suggested that POLN interacts with several proteins, including HELQ, Fanconi anemia (FA) core complex proteins, and FANCD2 (16). It is intriguing that this set of proteins has testis-related functions. Helq knock-out male mice have significantly smaller testes (14), targeted disruption of several FA genes caused impaired fertility in mice (39), and infertility is a common feature among male FA patients (40). To test these proposed interactions and to uncover possible molecular pathways relevant to POLN, we searched for proteins with the potential to associate with POLN in vivo. POLN was stably expressed as a FLAG and HA epitope fusion (ePOLN) in HeLa S3 cells. ePOLN was recovered from nuclear extracts by sequential immunoprecipitation with anti-FLAG and anti-HA antibodies (28, 29). The immunoprecipitate was separated on a gradient gel (Fig. 9A), and proteins from gel sections were identified by liquid chromatography-mass spectrometry. The data were filtered to eliminate common s. Among the resulting top 150 ranking hits there were nine DNA repair-related proteins (supplemental Table 1). Six of these (BRCA1, BRCA2, BARD1, PALB2, FANCJ, and RBBP8/CtIP) are components of the A, B, and C BRCA1-associated complexes related to homologous recombination (Fig. 9A and Table 3) (41). None of these proteins were present in a control FLAG-HA purification from HeLa S3 cells transfected with empty control vector. In parallel experiments in the same system using FLAG-HA-tagged HELQ as bait, none of these proteins were detected in the HELQ complex (13). Although FA core complex proteins FANCD2 and HELQ were previously proposed as POLN-interacting partners (16), no peptides representing any of these proteins were identified as ePOLN-associated proteins (supplemental Table 1), just as POLN was not detected in the HELQ complex in the previous study (13). Immunoblotting confirmed that BRCA1 was present in the immunoprecipitate and that FANCD2 and HELQ were present in the input fraction but undetectable in the POLN immunoprecipitate (Fig. 10A). To confirm POLN-BRCA1 and POLN-FANCJ interactions in another cell line, V5-tagged POLN was coexpressed with FLAG-HA-tagged BRCA1 or FANCJ in 293T cells. After immunoprecipitation from whole-cell extract with anti-FLAG and anti-HA antibodies, V5 antibody was used to identify POLN in the immunoprecipitate. Co-immunoprecipitation was observed between POLN and BRCA1 (Fig. 9B) and POLN and FANCJ (Fig. 9C).

Bottom Line: Consistent with this, injection of POLN-specific morpholino antisense oligonucleotides did not interfere with zebrafish embryonic development.These properties are conserved with the human enzyme.Although the physiological function of pol ν remains to be clarified, this study uncovers distinctive aspects of its expression control and evolutionarily conserved properties of this DNA polymerase.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Epigenetics and Molecular Carcinogenesis, University of Texas M.D. Anderson Cancer Center, Smithville, Texas 78957, the University of Texas Graduate School of Biomedical Sciences at Houston, Houston, Texas 77030, ktakata@mdanderson.org.

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Related in: MedlinePlus