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How a homolog of high-fidelity replicases conducts mutagenic DNA synthesis

Nature Structural & Molecular Biology volume 22, pages 298–303 (2015)Cite this article

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Abstract

All DNA replicases achieve high fidelity by a conserved mechanism, but each translesion polymerase carries out mutagenic DNA synthesis in its own way. Here we report crystal structures of human DNA polymerase ν (Pol ν), which is homologous to high-fidelity replicases yet is error prone. Instead of a simple open-to-closed movement of the O helix upon binding of a correct incoming nucleotide, Pol ν has a different open state and requires the finger domain to swing sideways and undergo both opening and closing motions to accommodate the nascent base pair. A single–amino acid substitution in the O helix of the finger domain improves the fidelity of Pol ν nearly ten-fold. A unique cavity and the flexibility of the thumb domain allow Pol ν to generate and accommodate a looped-out primer strand. Primer loop-out may be a mechanism for DNA trinucloetide-repeat expansion.

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Figure 1: Structure of Pol ν.
Figure 2: The unusual finger-open structure of Pol ν75.
Figure 3: Unique residues of Pol ν for mutagenic synthesis.
Figure 4: Primer looped out by Pol ν.
Figure 5: Diagram of DNA synthesis by Pol ν and a primer-loop-out model for TNR expansion.

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Acknowledgements

We thank D. Leahy and R. Craigie for editing the manuscript. The research was supported by the intramural research program of the US National Institutes of Health (DK036146-08, W.Y.).

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  1. Young-Sam Lee

    Present address: Present address: Well-Aging Research Center, Samsung Advanced Institute of Technology, Suwon, Korea.,

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  1. Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA

    Young-Sam Lee, Yang Gao & Wei Yang

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Contributions

Y.-S.L. carried out most of the experiments. Y.G. helped with kinetic measurement of K678A-mutant Pol ν and with data deposition. Y.-S.L. and W.Y. designed the project and prepared the manuscript.

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Correspondence to Wei Yang.

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Integrated supplementary information

Supplementary Figure 1 Cloning and characterization of Pol ν variants.

(a) Sequence alignment of Pol ν and the crystallized Klenow fragment of Bacillus Pol I. The first residues in Pol ν77, ν76, ν75 and ν74 are highlighted in green.

(b) Diagrams of expression constructs of four Pol ν variants. The His8 N-terminal to the two copies of maltose-binding protein (MBP) is not shown.

(c) PreScission protease digestion of Pol ν77, ν76, ν75 and ν74 after one-step purification with amylose resin. The results were analyzed by SDS gel.

(d) Size-exclusion chromatography analysis showed that Pol ν77 and DNA formed a stable complex of 1:1 molar ratio.

(e) Primer extension of the DNA substrate (with sequence shown) by tag-free Pol ν77, ν76 and ν75 (10 nM each) in the presence of all four nucleotides (dNTPs, 100µM total). Reactions were carried out with 100nM DNA in 20 mM Tris (pH 8.0), 80 mM NaCl, 1mM DTT, 10 mM MgCl2 and 0.1 mg/ml BSA at 37°C for 5 min. Pol ν75 was as active as Pol ν77.

Supplementary Figure 2 Purification and activity of WT and mutant Pol ν75.

(a) Results of Pol ν75 purification were shown in SDS gel. S200 stands for Superdex 200.

(b) Nucleotide selectivity. Four DNAs (100nM each) with varying templating nucleotide (A, G, T or C) were used to assay nucleotide selection by Pol ν77 in 10 µL of 20 mM Tris (pH 7.4), 50 mM NaCl, 0.1mg/ml BSA, 1 mM DTT, 5 mM MgCl2 and 100µM of dNTP at 37°C for 10 min.

(c) Purity of WT, E675R, K679A, and ΔIns2 Pol ν75 examined by SDS gel.

(d) The catalytic activities were assayed using a normal DNA substrate (sequence shown) as described in Methods (DNA synthesis) except for 5-min reaction time (not 10-min). E675R and ΔIns2 had reduced activity.

Supplementary Figure 3 Diagram of DNA sequences used in crystallizing Pol ν75.

(a) DNA in the Ndna1 crystal. The designed and actual DNA crystallized are shown. Pol ν75 is shown as a Packman.

(b) The SAD-MR experimental electron density map is contoured at 1.0 σ in pea green. The 4 Br sites are obvious based on the anomalous peaks contoured at 8.0 σ in blue.

(c) DNAs designed for Ndna2 and Ndna3 are shown side-by-side. The 2Fo-Fc (1.0 σ) and Fo-Fc map (3.0 σ) corresponding to the DNAs are shown below. In Ndna2 the 4-nt overhang formed 5 bps overhang at a crystal dyad instead of 4 bps as in Ndna3, which was explained by a misalignment and gap-filling by dAMPNPP (a non-hydrolyzable analog of incoming nucleotide) as diagramed.

(d) DNA in Ndna4. The designed and actual DNA crystallized are shown side-by-side. The overhang at the crystal dyad also formed 5 bps as in Ndna2.

Supplementary Figure 4 Comparison of Pol ν75 and E. coli Klenow fragment on looped-out primer DNA.

(a) Sequences and structures of normal (fully base paired, Full-bp or F-bp) DNA and seven variations with looped-out nucleotide in primer.

(b) Primer extension assay of Pol ν75 (40 nM) and the E. coli Klenow fragment (New England BioLabs, 0.0357 unit) with different DNA substrates in the presence of each dNTP (A, G, T or C) or all four dNTPs (4). Reactions were carried out as described in Methods (DNA synthesis) except for pH 7.4 (not pH 8.3) and 5-min reaction time (not 10-min). Pol ν75 was active with P[3,1] and P[4 or 5,1], while Klenow had weak activity with P[4 or 5,1].

Supplementary Figure 5 Cloning, purification and activity of Pol θ90 and Pol θ86.

(a) According to the secondary structure prediction and the previously published active Pol θ90 construct, the cloning site of Pol θ86 was chosen to remove predicted additional random coils (indicated as C)

(b) Expression, purification and PreScission cleavage of 2MBP tagged Pol θ90 and Pol θ86. Results are shown in SDS gel. The 2MBP tag and tag-free Pol θ90 and Pol θ86 have similar sizes and overlap on the SDS gel.

(c) Pol θ86 was purified as Pol ν75. The elution points from Heparin and Superdex 200 (S200) of the two proteins differed.

(d) Pol θ90 and Pol θ86 were equally active in DNA synthesis, before or after PreScission cleavage.

(e) Activity comparison of Pol ν75 and Pol θ86. The specific activity of Pol ν75 is roughly one fourth of that of Pol θ86. Reactions were carried out as described in Methods (DNA synthesis).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Tables 1 and 2 (PDF 1983 kb)

Finger movement between the conventional open and closed state exemplified by bacillus Pol I

The binary complex (4BDP) and ternary complex (3THV) were compared. The three domains of the catalytic core are color-coded and the Exo domain is shown in silver. The M, N (lilac) and O (purple) helices undergo the large closing movement (40° rotation). The Oa and Ob helices move slightly towards the N and O helices. (MOV 1962 kb)

Conformational changes of Pol ν from the unusual open (Ndna3) to the closed state (Ndna2).

The Oa and Ob helices are colored in light blue. The remaining regions are colored as in movie 1. The M helix is disordered in Pol ν. The N (lilac) and O (purple) helices undergo a closing movement towards the DNA (25° rotation). The Oa and Ob helices move outwards by a 17° rotation. (MOV 1936 kb)

The 30° rotation of the thumb domain in Pol ν

The conformational changes between Ndna2 (closed finger and thumb-in) and Ndna4 (closed finger and thumb-out) structure. Y686, shown in stick-and-ball model, is a placeholder for the incoming templating base. (MOV 1736 kb)

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Lee, YS., Gao, Y. & Yang, W. How a homolog of high-fidelity replicases conducts mutagenic DNA synthesis. Nat Struct Mol Biol 22, 298–303 (2015). https://doi.org/10.1038/nsmb.2985

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