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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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DNA polymerase activity of zebrafish POLN (DrPOLN).A, constructs containing residues 276–1146 of DrPOLN were bacterially expressed and purified. Substituted residues in DrPOLN derivatives (Asp-902 and Arg-957) are shown in Fig. 1. Three hundred ng of purified DrPOLN derivatives and molecular mass markers were separated by electrophoresis in a 4–15% SDS-polyacrylamide gradient gel and stained with colloidal Coomassie Brilliant Blue G-250. B, DNA polymerase activities of DrPOLN. 23 nm DrPOLN and DrPOLN (D902A) and 10 pm RB69 gp43 were incubated with the 5′-32P-labeled primer-templates indicated under “Experimental Procedures” in the presence of all four dNTPs at 37 °C for the indicated time. The activities were analyzed on the same gel.
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Figure 6: DNA polymerase activity of zebrafish POLN (DrPOLN).A, constructs containing residues 276–1146 of DrPOLN were bacterially expressed and purified. Substituted residues in DrPOLN derivatives (Asp-902 and Arg-957) are shown in Fig. 1. Three hundred ng of purified DrPOLN derivatives and molecular mass markers were separated by electrophoresis in a 4–15% SDS-polyacrylamide gradient gel and stained with colloidal Coomassie Brilliant Blue G-250. B, DNA polymerase activities of DrPOLN. 23 nm DrPOLN and DrPOLN (D902A) and 10 pm RB69 gp43 were incubated with the 5′-32P-labeled primer-templates indicated under “Experimental Procedures” in the presence of all four dNTPs at 37 °C for the indicated time. The activities were analyzed on the same gel.

Mentions: The assays were performed as reported (3, 4). The 5′-32P-labeled primers (5′-CACTGACTGTATGA-3′ or 5′-CACTGACTGTATGAT-3′) in Fig. 7 are similar but are two or one nucleotide shorter than the primer used for DNA polymerase assays in Fig. 6 (5′-CACTGACTGTATGATG-3′). The 5′-32P-labeled 14- or 15-mer primer and a 30-mer template (sequences given above) were annealed at a molar ratio of 1:1. Primer extension reactions with DrPOLN and R957A were as described above. 10 μl of reaction mixtures were incubated at 37 °C for 2, 4, and 6 min and diluted in 10 μl of formamide stop buffer. Products were heated at 95 °C for 3 min and separated on a denaturing 20% polyacrylamide, 7 m urea gel. Product bands were quantified by PhosphorImager, and the values were used to calculate the probability of termination of processive synthesis and the insertion efficiencies at each template nucleotide. The termination probability at any position (N) is defined as the band intensity at N divided by the total intensity for all bands ≥N. The insertion probability at any position (N) is defined as the intensity at bands ≥N divided by the intensity at bands ≥N − 1. The extension probability at any position (N) is defined as the band intensity ≥N + 1 divided by the intensity at bands ≥N. The bypass probability at position N is defined as the band density ≥N + 1 divided by the intensity of ≥N1. To detect the bypass efficiency, the bypass probability (damaged) is divided by the bypass probability (undamaged) as described (25). The values are averages from two independent experiments at reaction intervals from 2, 4, and 6 min.


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)

DNA polymerase activity of zebrafish POLN (DrPOLN).A, constructs containing residues 276–1146 of DrPOLN were bacterially expressed and purified. Substituted residues in DrPOLN derivatives (Asp-902 and Arg-957) are shown in Fig. 1. Three hundred ng of purified DrPOLN derivatives and molecular mass markers were separated by electrophoresis in a 4–15% SDS-polyacrylamide gradient gel and stained with colloidal Coomassie Brilliant Blue G-250. B, DNA polymerase activities of DrPOLN. 23 nm DrPOLN and DrPOLN (D902A) and 10 pm RB69 gp43 were incubated with the 5′-32P-labeled primer-templates indicated under “Experimental Procedures” in the presence of all four dNTPs at 37 °C for the indicated time. The activities were analyzed on the same gel.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4591814&req=5

Figure 6: DNA polymerase activity of zebrafish POLN (DrPOLN).A, constructs containing residues 276–1146 of DrPOLN were bacterially expressed and purified. Substituted residues in DrPOLN derivatives (Asp-902 and Arg-957) are shown in Fig. 1. Three hundred ng of purified DrPOLN derivatives and molecular mass markers were separated by electrophoresis in a 4–15% SDS-polyacrylamide gradient gel and stained with colloidal Coomassie Brilliant Blue G-250. B, DNA polymerase activities of DrPOLN. 23 nm DrPOLN and DrPOLN (D902A) and 10 pm RB69 gp43 were incubated with the 5′-32P-labeled primer-templates indicated under “Experimental Procedures” in the presence of all four dNTPs at 37 °C for the indicated time. The activities were analyzed on the same gel.
Mentions: The assays were performed as reported (3, 4). The 5′-32P-labeled primers (5′-CACTGACTGTATGA-3′ or 5′-CACTGACTGTATGAT-3′) in Fig. 7 are similar but are two or one nucleotide shorter than the primer used for DNA polymerase assays in Fig. 6 (5′-CACTGACTGTATGATG-3′). The 5′-32P-labeled 14- or 15-mer primer and a 30-mer template (sequences given above) were annealed at a molar ratio of 1:1. Primer extension reactions with DrPOLN and R957A were as described above. 10 μl of reaction mixtures were incubated at 37 °C for 2, 4, and 6 min and diluted in 10 μl of formamide stop buffer. Products were heated at 95 °C for 3 min and separated on a denaturing 20% polyacrylamide, 7 m urea gel. Product bands were quantified by PhosphorImager, and the values were used to calculate the probability of termination of processive synthesis and the insertion efficiencies at each template nucleotide. The termination probability at any position (N) is defined as the band intensity at N divided by the total intensity for all bands ≥N. The insertion probability at any position (N) is defined as the intensity at bands ≥N divided by the intensity at bands ≥N − 1. The extension probability at any position (N) is defined as the band intensity ≥N + 1 divided by the intensity at bands ≥N. The bypass probability at position N is defined as the band density ≥N + 1 divided by the intensity of ≥N1. To detect the bypass efficiency, the bypass probability (damaged) is divided by the bypass probability (undamaged) as described (25). The values are averages from two independent experiments at reaction intervals from 2, 4, and 6 min.

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.

Show MeSH
Related in: MedlinePlus