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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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Evolutionarily conserved residue is important for translesion synthesis activity in DrPOLN.A, 23 nm DrPOLN and DrPOLN (R957A), denoted as WT and R957A, respectively, were incubated with the 5′-32P-labeled primer-templates indicated above the panel; DNA synthesis on a DNA template containing an undamaged thymine (lanes 1–16) or a 5S-Tg (lanes 17–32) from the 14-mer primer (lanes 1–8 and 17–24) or the 15-mer primer (lanes 9–16 and 25–32) is shown. All reaction mixtures contained substrate at 100 nm in the presence of all four deoxynucleotide triphosphates. Incubation time of each reaction is shown at bottom. Locations of unreacted end-labeled primer (N0), each template base position (from N1 to N16), full-length product (N16 for the 14-mer primer and N15 for the 15-mer primer), and positions of 5S-Tg are shown as Tg.
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Figure 7: Evolutionarily conserved residue is important for translesion synthesis activity in DrPOLN.A, 23 nm DrPOLN and DrPOLN (R957A), denoted as WT and R957A, respectively, were incubated with the 5′-32P-labeled primer-templates indicated above the panel; DNA synthesis on a DNA template containing an undamaged thymine (lanes 1–16) or a 5S-Tg (lanes 17–32) from the 14-mer primer (lanes 1–8 and 17–24) or the 15-mer primer (lanes 9–16 and 25–32) is shown. All reaction mixtures contained substrate at 100 nm in the presence of all four deoxynucleotide triphosphates. Incubation time of each reaction is shown at bottom. Locations of unreacted end-labeled primer (N0), each template base position (from N1 to N16), full-length product (N16 for the 14-mer primer and N15 for the 15-mer primer), and positions of 5S-Tg are shown as Tg.

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)

Evolutionarily conserved residue is important for translesion synthesis activity in DrPOLN.A, 23 nm DrPOLN and DrPOLN (R957A), denoted as WT and R957A, respectively, were incubated with the 5′-32P-labeled primer-templates indicated above the panel; DNA synthesis on a DNA template containing an undamaged thymine (lanes 1–16) or a 5S-Tg (lanes 17–32) from the 14-mer primer (lanes 1–8 and 17–24) or the 15-mer primer (lanes 9–16 and 25–32) is shown. All reaction mixtures contained substrate at 100 nm in the presence of all four deoxynucleotide triphosphates. Incubation time of each reaction is shown at bottom. Locations of unreacted end-labeled primer (N0), each template base position (from N1 to N16), full-length product (N16 for the 14-mer primer and N15 for the 15-mer primer), and positions of 5S-Tg are shown as Tg.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Evolutionarily conserved residue is important for translesion synthesis activity in DrPOLN.A, 23 nm DrPOLN and DrPOLN (R957A), denoted as WT and R957A, respectively, were incubated with the 5′-32P-labeled primer-templates indicated above the panel; DNA synthesis on a DNA template containing an undamaged thymine (lanes 1–16) or a 5S-Tg (lanes 17–32) from the 14-mer primer (lanes 1–8 and 17–24) or the 15-mer primer (lanes 9–16 and 25–32) is shown. All reaction mixtures contained substrate at 100 nm in the presence of all four deoxynucleotide triphosphates. Incubation time of each reaction is shown at bottom. Locations of unreacted end-labeled primer (N0), each template base position (from N1 to N16), full-length product (N16 for the 14-mer primer and N15 for the 15-mer primer), and positions of 5S-Tg are shown as Tg.
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