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Comparative Study
. 2004 Nov 10;23(22):4484-94.
doi: 10.1038/sj.emboj.7600424. Epub 2004 Oct 21.

High-efficiency Bypass of DNA Damage by Human DNA Polymerase Q

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Free PMC article
Comparative Study

High-efficiency Bypass of DNA Damage by Human DNA Polymerase Q

Mineaki Seki et al. EMBO J. .
Free PMC article

Abstract

Endogenous DNA damage arises frequently, particularly apurinic (AP) sites. These must be dealt with by cells in order to avoid genotoxic effects. DNA polymerase theta; is a newly identified enzyme encoded by the human POLQ gene. We find that POLQ has an exceptional ability to bypass an AP site, inserting A with 22% of the efficiency of a normal template, and continuing extension as avidly as with a normally paired base. POLQ preferentially incorporates A opposite an AP site and strongly disfavors C. On nondamaged templates, POLQ makes frequent errors, incorporating G or T opposite T about 1% of the time. This very low fidelity distinguishes POLQ from other A-family polymerases. POLQ has three sequence insertions between conserved motifs in its catalytic site. One insert of approximately 22 residues into the tip of the polymerase thumb subdomain is predicted to confer considerable flexibility and additional DNA contacts to affect enzyme fidelity. POLQ is the only known enzyme that efficiently carries out both the insertion and extension steps for bypass of AP sites, commonly formed as endogenous genomic lesions.

Figures

Figure 1
Figure 1
POLQ is an error-prone enzyme. (A) Full-length recombinant human POLQ was purified as described and 75 ng was electrophoresed on an SDS–polyacrylamide gel and silver-stained (lane 2). Molecular weight markers are shown in lane 1. (B) Template used for the primer extension assay. 5′-32P-labeled 16-mer was annealed to 30-mer DNA. The first template base is denoted by X. (C) POLQ frequently misincorporates residues. The first template base (X) was changed from T (lanes 1–4) to A (lanes 5–8), G (lanes 9–12), and C (lanes 13–16). Template bases are indicated on the left.
Figure 2
Figure 2
Nucleotide preference for bypass of an AP site. A 5′-32P-labeled 16-mer primer was annealed to (A) 30-mer template DNA or (B) a template containing an AP site. Either all four dNTPs (N4) or each deoxynucleotide separately at 100 μM was utilized in reactions as indicated.
Figure 3
Figure 3
POLQ bypasses AP sites and Tg. (A–G) A 5′-32P-labeled 16-mer was annealed with 30-mer template DNA containing lesions and incubated with either human POLQ (Q), exonuclease-defective pol I Klenow fragment (K), or pol η (η). (A) Undamaged control, (B) AP analog, (C) 5R-Tg, (D) 5S-Tg, (E) T–T CPD, (F) T–T 6-4 PP, and (G) 1,2-d(GpG) cisplatin adduct. The position of the adduct is indicated by an arrow (B–D) or bracket (E–G).
Figure 4
Figure 4
Incorporation opposite an AP site takes place directly rather than by template slippage. A 5′-[32P]-labeled 16-mer was annealed to 30-mer templates containing an AP site at the next template position as in Figures 2A and 3A. Four templates were used, having either T, A, G, or C as the next template base following the AP site. Each of the four possible reaction mixtures containing only a single dNTP was tested. After a 10 min incubation at 37°C, an A was preferentially incorporated opposite the AP site, regardless of the following sequence context.
Figure 5
Figure 5
Sequence insertions in the catalytic site of POLQ. Sequence alignment of the polymerase catalytic region of POLQ/Mus308 family members and other prokaryotic A-family polymerases is shown. Identical amino acids are indicated with dark shading and similar amino acids with light shading. The alignment was carried out using the Clustal X program. The locations of the inserts and the conserved DNA polymerase motifs 1–6 are indicated. A putative PCNA binding motif is denoted by asterisks. EcPolI: E. coli pol I; TaqPolI: Thermus aquaticus pol I; BstPolI: Bacillus stearothermophilus pol I; Anaero: Anaerocellum thermophilum pol I; Ricketts: Rickettsia prowazekii pol I; Haein: Haemophilus influenzae pol I; Trepon: Treponema pallidum pol I; Deinorad: Deinococcus radiodurans pol I; Rhod: Rhodothermus marinus pol I; Myctu: Mycobacterium tuberculosis pol I; Helico: Helicobacter pylori pol I; HsPOLQ: human POLQ; MsPOLQ: mouse POLQ; hsPOLN: human POLN; mmPOLN: mouse POLN; Mus308: Drosophila melanogaster Mus308; CeMus1: Caenorhabditis elegans Mus1.
Figure 6
Figure 6
Sequence insertions in the catalytic site of POLQ. (A) The DNA polymerase catalytic region of POLQ is shown in relation to the other A-family polymerases human POLN, Drosophila Mus308, and Taq pol I. The locations of the inserts are shown in red and the conserved DNA polymerase motifs 1–6 are indicated as gray regions. Nonconserved differences in the insert regions are indicated in green. The bottom line shows coding regions for the main finger, palm, and thumb structural domains. (B, C) A computationally derived model for the polymerase domain of POLQ–DNA (B) obtained with the Amber94 potential, as compared to the known structure of the closed form of Taq pol I (C). The polymerase domain of Taq pol I (1QTM) is shown in white, while the modeled closed structure of human POLQ is colored blue to red, in the increasing order of r.m.s. deviation from Taq pol I. In both diagrams, the DNA template strand is green, and the primer strand is red. Gray spheres show the location of two Mg2+ cations, and an incoming ddTTP is positioned nearby in the structure. Inserts 1–3 of POLQ are shown in a ball-and-stick mode. The model is not meant to propose a detailed structure for the inserts, but instead to provide insight into the potential of Insert 1 to make contact with template DNA.

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