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. 2010 Jun;38(10):3233-44.
doi: 10.1093/nar/gkq048. Epub 2010 Feb 9.

Evolutionary conservation of residues in vertebrate DNA polymerase N conferring low fidelity and bypass activity

Kei-ichi Takata  1 , Mercedes E Arana, Mineaki Seki, Thomas A Kunkel, Richard D Wood
Affiliations

Evolutionary conservation of residues in vertebrate DNA polymerase N conferring low fidelity and bypass activity (VSports)

V体育2025版 - Kei-ichi Takata et al. Nucleic Acids Res. 2010 Jun.

"VSports" Abstract

POLN is a nuclear A-family DNA polymerase encoded in vertebrate genomes. POLN has unusual fidelity and DNA lesion bypass properties, including strong strand displacement activity, low fidelity favoring incorporation of T for template G and accurate translesion synthesis past a 5S-thymine glycol (5S-Tg) VSports手机版. We searched for conserved features of the polymerase domain that distinguish it from prokaryotic pol I-type DNA polymerases. A Lys residue (679 in human POLN) of particular interest was identified in the conserved 'O-helix' of motif 4 in the fingers sub-domain. The corresponding residue is one of the most important for controlling fidelity of prokaryotic pol I and is a nonpolar Ala or Thr in those enzymes. Kinetic measurements show that K679A or K679T POLN mutant DNA polymerases have full activity on nondamaged templates, but poorly incorporate T opposite template G and do not bypass 5S-Tg efficiently. We also found that a conserved Tyr residue in the same motif not only affects sensitivity to dideoxynucleotides, but also greatly influences enzyme activity, fidelity and bypass. Protein sequence alignment reveals that POLN has three specific insertions in the DNA polymerase domain. The results demonstrate that residues have been strictly retained during evolution that confer unique bypass and fidelity properties on POLN. .

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Figures (V体育ios版)

Figure 1.
Figure 1.
Distinct sequence insertions in POLN. (A) The DNA polymerase catalytic region of human POLN is shown in relation to Taq pol and human POLQ. The locations of the three inserts found in POLN and POLQ are shown in red and the conserved DNA polymerase motifs are shown in gray. (B) Sequence alignment of motif 4 of POLN, POLQ and prokaryotic A-family DNA polymerases. Similarity groups for colored residues are (K, R, H), (D, E), (F, Y, W) and (Q, N). Perfectly conserved residues are denoted by ++, relatively conserved residues are denoted by +, flexible amino acid residues are denoted by ∨. K679 and Y682 are shown in red and green in a modeled structure for motif 4 of HsPOLN. Hs, Homo sapiens; Pt, Pan troglodytes; Mm, Mus musculus; Cf, Canis familiaris; Gg, Gallus gallus; Dr, Danio rerio; Mus308, Drosophila melanogaster Mus308; CeMus1, Caenorhabditis elegans Mus1; Taqpol, Thermus aquaticus DNA polymerase; EcpolI, Escherichia coli; BspolI, Bacillus subtilis; Myctu, Mycobacterium tuberculosis pol I; Haein, Haemophilus influenzae pol I; Ricketts, Rickettsia prowazekii pol I; Trepon, Treponema pallidum pol I; T7pol, T7 DNA polymerase; T5pol, T5 DNA polymerase. PtPOLN, CfPOLN and GgPOLN are predicted from their genomic DNA sequences.
Figure 2.
Figure 2.
Activity of wild-type (WT) and mutant POLN on a nondamaged DNA template. (A) Purified POLN derivatives (300 ng) and molecular weight markers were separated by electrophoresis in a 4–15% SDS–polyacrylamide gradient gel and stained with colloidal Coomassie Brilliant Blue G-250. Substituted residues in POLN derivatives are shown in Figure 1. delP is POLN with a short truncation of the C-terminal proline-rich tail. (B) DNA polymerase activities of POLN derivatives. Increasing amounts of delP, WT, K679R, K679T, K679A and K679Q (6, 12 and 23 nM), Y682F (6, 12, 23, 29, 58 and 115 nM), POLQ (3, 6 and 12 nM), RB69 gp43 (2.5, 5 and 10 pM) were incubated with the 5′-32P-labeled primer templates indicated beside the panel in the presence of all 4 nt at 37°C for 10 min. The first lane contained no enzyme. The percentage (%) of the product extension from the primer is shown below each lane. The specific activity of POLN is 127 U/mg, delP is 125 U/mg, K679R is 116 U/mg, K679T is 112 U/mg, K679A is 124 U/mg, K679Q is 153 U/mg and Y682F is 32 U/mg. One unit is defined as 10 nmol of dTTP incorporated into poly(dA)/oligo(dT)10:1 template at 37°C for 30 min.
Figure 3.
Figure 3.
Substitution of Phe for Tyr in POLN increases discrimination against dideoxynucleotide. Escherichia coli Kf (exo−), delP, WT and Y682F were incubated with ddTTP in a reaction mixture containing radioactively labeled dTTP and poly(dA)/oligo(dT) template.
Figure 4.
Figure 4.
Nucleotide selectivities of POLN derivatives. Twenty-three nanomolars of delP, WT, K679R, K679T, K679A and K679Q, 115 nM of Y682F, 12 nM of POLQ and 10 pM of RB69 gp43 were incubated with 300 fmol of 5′-32P-labeled 16-mer primer annealed to a 30-mer DNA template in the presence of one of the indicated dNTPs (100 µM) for 10 min. The first template base denoted by X was G, T, A or C in (A, B, C and D), respectively. Template sequences are indicated to the left of each panel.
Figure 5.
Figure 5.
Translesion synthesis activities of POLN derivatives. Increasing amounts of delP, WT, K679R, K679T, K679A and K679Q (6, 12 and 23 nM), Y682F (6, 12, 23, 29, 58 and 115 nM), POLQ (3, 6 and 12 nM) and RB69 gp43 (2.5, 5 and 10 pM) were incubated with the 5′-32P-labeled primer templates indicated, in the presence of all four dNTPs for 10 min. The first lane contained no enzyme. The first base templates were 5S-Tg (A), 5R-Tg (B) or AP analog (C). The percentage (%) extension of the primer is shown below each lane.
Figure 6.
Figure 6.
Mutation of Lys 679 to Ala or Thr reduces strand displacement. Increasing amounts of delP, WT, K679R, K679T, K679A and K679Q (12, 23 and 46 nM), and Y682F (12, 23, 46, 58, 115 and 230 nM) and RB69 gp43 (5, 10, 20, 103, 205 and 410 pM) were incubated at 37°C for 15 min with 100 fmol primer annealed to M13mp18GTGx (A) or nicked substrate, 100 fmol primer and downstream oligomer were annealed to M13mp18GTGx (B). The substrates are schematically diagrammed and were radiolabeled at the 5′-end of the upstream 24-mer primer (asterisk). The 5′-end of the downstream 60-mer oligonucleotide was phosphorylated, and its 3′-end was blocked with ddATP. The first lane contained no enzyme. The percentage (%) extension of the primer is shown below each lane.
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References

    1. Takata K, Shimizu T, Iwai S, Wood RD. Human DNA polymerase N (POLN) is a low-fidelity enzyme capable of error-free bypass of 5S-thymine glycol. J. Biol. Chem. 2006;281:23445–23455. - PubMed (VSports注册入口)
    1. Arana ME, Takata K, Garcia-Diaz M, Wood RD, Kunkel TA. A unique error signature for human DNA polymerase ν. DNA Repair. 2007;6:213–223. - PMC (VSports最新版本) - PubMed
    1. Seki M, Marini F, Wood RD. POLQ (Pol θ), a DNA polymerase and DNA-dependent ATPase in human cells. Nucleic Acids Res. 2003;31:6117–6126. - PMC - PubMed
    1. Arana ME, Seki M, Wood RD, Rogozin IB, Kunkel TA. Low-fidelity DNA synthesis by human DNA polymerase theta. Nucleic Acids Res. 2008;36:3847–3856. - V体育官网入口 - PMC - PubMed
    1. Shima N, Munroe RJ, Schimenti JC. The mouse genomic instability mutation chaos1 is an allele of Polq that exhibits genetic interaction with Atm. Mol. Cell Biol. 2004;24:10381–10389. - PMC - PubMed

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