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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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Nucleotide selectivities of DrPOLN and HsPOLN derivatives.A, 23 nm DrPOLN and DrPOLN (R957A), denoted as wild-type and R957A, respectively, were incubated with 300 fmol of 5′-32P-labeled 16-mer primer annealed to a 30-mer DNA template in the presence of four or one of the indicated dNTPs (100 μm) for 10 min. The first template base denoted by X was G or T. Template sequences are indicated above the panel. NE indicates no enzyme. B, as described for A, using HsPOLN and HsPOLN (K679A), denoted as wild-type and K679A, respectively. C, 23 nm DrPOLN and DrPOLN (R957A), denoted as wild-type and R957A, respectively, were incubated with 5′-32P-labeled 16-mer primer annealed to a 30-mer DNA template, in which the first template base was G in the presence of all four dNTPs or dTTP (100 μm) for 10 min in indicated pH conditions. D, as described for C, using HsPOLN and HsPOLN (K679A), denoted as wild-type and K679A, respectively. The percentage (%) of the product extension from the primer is shown below each lane in C and D.
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Figure 8: Nucleotide selectivities of DrPOLN and HsPOLN derivatives.A, 23 nm DrPOLN and DrPOLN (R957A), denoted as wild-type and R957A, respectively, were incubated with 300 fmol of 5′-32P-labeled 16-mer primer annealed to a 30-mer DNA template in the presence of four or one of the indicated dNTPs (100 μm) for 10 min. The first template base denoted by X was G or T. Template sequences are indicated above the panel. NE indicates no enzyme. B, as described for A, using HsPOLN and HsPOLN (K679A), denoted as wild-type and K679A, respectively. C, 23 nm DrPOLN and DrPOLN (R957A), denoted as wild-type and R957A, respectively, were incubated with 5′-32P-labeled 16-mer primer annealed to a 30-mer DNA template, in which the first template base was G in the presence of all four dNTPs or dTTP (100 μm) for 10 min in indicated pH conditions. D, as described for C, using HsPOLN and HsPOLN (K679A), denoted as wild-type and K679A, respectively. The percentage (%) of the product extension from the primer is shown below each lane in C and D.

Mentions: Low fidelity favoring incorporation of T for template G is a biochemical property of HsPOLN (3–5). However, this tendency was not evident in DrPOLN (Fig. 8). Wild-type HsPOLN but not the K679A or K679T HsPOLN mutants efficiently incorporated T for G at the optimal pH of 8.8 (3). When the pH was reduced to 8.0 or 7.2, the DNA polymerase activity and the T misincorporation activity of HsPOLN were both reduced as reported (Fig. 8, B and D) (23). Unlike HsPOLN, wild-type DrPOLN did not efficiently incorporate T opposite template G even at pH 8.8; the Arg residue at 957 did not influence T or G misincorporation (Fig. 8, A and C). In addition, DrPOLN efficiently incorporated G opposite G unlike HsPOLN (Fig. 8, A and B).


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)

Nucleotide selectivities of DrPOLN and HsPOLN derivatives.A, 23 nm DrPOLN and DrPOLN (R957A), denoted as wild-type and R957A, respectively, were incubated with 300 fmol of 5′-32P-labeled 16-mer primer annealed to a 30-mer DNA template in the presence of four or one of the indicated dNTPs (100 μm) for 10 min. The first template base denoted by X was G or T. Template sequences are indicated above the panel. NE indicates no enzyme. B, as described for A, using HsPOLN and HsPOLN (K679A), denoted as wild-type and K679A, respectively. C, 23 nm DrPOLN and DrPOLN (R957A), denoted as wild-type and R957A, respectively, were incubated with 5′-32P-labeled 16-mer primer annealed to a 30-mer DNA template, in which the first template base was G in the presence of all four dNTPs or dTTP (100 μm) for 10 min in indicated pH conditions. D, as described for C, using HsPOLN and HsPOLN (K679A), denoted as wild-type and K679A, respectively. The percentage (%) of the product extension from the primer is shown below each lane in C and D.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 8: Nucleotide selectivities of DrPOLN and HsPOLN derivatives.A, 23 nm DrPOLN and DrPOLN (R957A), denoted as wild-type and R957A, respectively, were incubated with 300 fmol of 5′-32P-labeled 16-mer primer annealed to a 30-mer DNA template in the presence of four or one of the indicated dNTPs (100 μm) for 10 min. The first template base denoted by X was G or T. Template sequences are indicated above the panel. NE indicates no enzyme. B, as described for A, using HsPOLN and HsPOLN (K679A), denoted as wild-type and K679A, respectively. C, 23 nm DrPOLN and DrPOLN (R957A), denoted as wild-type and R957A, respectively, were incubated with 5′-32P-labeled 16-mer primer annealed to a 30-mer DNA template, in which the first template base was G in the presence of all four dNTPs or dTTP (100 μm) for 10 min in indicated pH conditions. D, as described for C, using HsPOLN and HsPOLN (K679A), denoted as wild-type and K679A, respectively. The percentage (%) of the product extension from the primer is shown below each lane in C and D.
Mentions: Low fidelity favoring incorporation of T for template G is a biochemical property of HsPOLN (3–5). However, this tendency was not evident in DrPOLN (Fig. 8). Wild-type HsPOLN but not the K679A or K679T HsPOLN mutants efficiently incorporated T for G at the optimal pH of 8.8 (3). When the pH was reduced to 8.0 or 7.2, the DNA polymerase activity and the T misincorporation activity of HsPOLN were both reduced as reported (Fig. 8, B and D) (23). Unlike HsPOLN, wild-type DrPOLN did not efficiently incorporate T opposite template G even at pH 8.8; the Arg residue at 957 did not influence T or G misincorporation (Fig. 8, A and C). In addition, DrPOLN efficiently incorporated G opposite G unlike HsPOLN (Fig. 8, A and B).

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