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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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POLN is associated with BRCA1, FANCJ, and other homologous recombination components.A, POLN-associated proteins were immunopurified from nuclear extracts prepared from HeLa S3 cells expressing FLAG-HA epitope-tagged POLN. The complex was sequentially purified with anti-FLAG and anti-HA antibodies, resolved by SDS-PAGE on a 4–20% gradient gel, and visualized by silver staining. Approximate migration positions of proteins identified in gel sections are shown. B, V5-tagged POLN and FLAG-HA epitope-tagged BRCA1 were transiently co-expressed in 293T cells. The whole-cell extracts prepared from the transfected cells were sonicated and incubated in the presence of benzonase. FLAG-HA epitope-tagged BRCA1 was immunoprecipitated (IP) with anti-FLAG and anti-HA antibodies. V5-tagged POLN in the immunoprecipitated samples was detected with anti-V5 antibody. C, interaction between V5-tagged POLN and FLAG-HA epitope-tagged FANCJ was examined similarly as described in B.
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Figure 9: POLN is associated with BRCA1, FANCJ, and other homologous recombination components.A, POLN-associated proteins were immunopurified from nuclear extracts prepared from HeLa S3 cells expressing FLAG-HA epitope-tagged POLN. The complex was sequentially purified with anti-FLAG and anti-HA antibodies, resolved by SDS-PAGE on a 4–20% gradient gel, and visualized by silver staining. Approximate migration positions of proteins identified in gel sections are shown. B, V5-tagged POLN and FLAG-HA epitope-tagged BRCA1 were transiently co-expressed in 293T cells. The whole-cell extracts prepared from the transfected cells were sonicated and incubated in the presence of benzonase. FLAG-HA epitope-tagged BRCA1 was immunoprecipitated (IP) with anti-FLAG and anti-HA antibodies. V5-tagged POLN in the immunoprecipitated samples was detected with anti-V5 antibody. C, interaction between V5-tagged POLN and FLAG-HA epitope-tagged FANCJ was examined similarly as described in B.

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)

POLN is associated with BRCA1, FANCJ, and other homologous recombination components.A, POLN-associated proteins were immunopurified from nuclear extracts prepared from HeLa S3 cells expressing FLAG-HA epitope-tagged POLN. The complex was sequentially purified with anti-FLAG and anti-HA antibodies, resolved by SDS-PAGE on a 4–20% gradient gel, and visualized by silver staining. Approximate migration positions of proteins identified in gel sections are shown. B, V5-tagged POLN and FLAG-HA epitope-tagged BRCA1 were transiently co-expressed in 293T cells. The whole-cell extracts prepared from the transfected cells were sonicated and incubated in the presence of benzonase. FLAG-HA epitope-tagged BRCA1 was immunoprecipitated (IP) with anti-FLAG and anti-HA antibodies. V5-tagged POLN in the immunoprecipitated samples was detected with anti-V5 antibody. C, interaction between V5-tagged POLN and FLAG-HA epitope-tagged FANCJ was examined similarly as described in B.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 9: POLN is associated with BRCA1, FANCJ, and other homologous recombination components.A, POLN-associated proteins were immunopurified from nuclear extracts prepared from HeLa S3 cells expressing FLAG-HA epitope-tagged POLN. The complex was sequentially purified with anti-FLAG and anti-HA antibodies, resolved by SDS-PAGE on a 4–20% gradient gel, and visualized by silver staining. Approximate migration positions of proteins identified in gel sections are shown. B, V5-tagged POLN and FLAG-HA epitope-tagged BRCA1 were transiently co-expressed in 293T cells. The whole-cell extracts prepared from the transfected cells were sonicated and incubated in the presence of benzonase. FLAG-HA epitope-tagged BRCA1 was immunoprecipitated (IP) with anti-FLAG and anti-HA antibodies. V5-tagged POLN in the immunoprecipitated samples was detected with anti-V5 antibody. C, interaction between V5-tagged POLN and FLAG-HA epitope-tagged FANCJ was examined similarly as described in B.
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