DNA polymerase theta (POLQ) can extend from mismatches and from bases opposite a (6-4) photoproduct

Abstract

DNA polymerase theta (pol theta) is a nuclear A-family DNA polymerase encoded by the POLQ gene in vertebrate cells. The biochemical properties of pol theta and of Polq-defective mice have suggested that pol theta participates in DNA damage tolerance. For example, pol theta was previously found to be proficient not only in incorporation of a nucleotide opposite a thymine glycol or an abasic site, but also extends a polynucleotide chain efficiently from the base opposite the lesion. We carried out experiments to determine whether this ability to extend from non-standard termini is a more general property of the enzyme. Pol theta extended relatively efficiently from matched termini as well as termini with A:G, A:T and A:C mismatches, with less descrimination than a well-studied A-family DNA polymerase, exonuclease-free pol I from E. coli. Although pol theta was unable to, by itself, bypass a cyclobutane pyrimidine dimer or a (6-4) photoproduct, it could perform some extension from primers with bases placed across from these lesions. When pol theta was combined with DNA polymerase iota, an enzyme that can insert a base opposite a UV-induced (6-4) photoproduct, complete bypass of a (6-4) photoproduct was possible. These data show that in addition to its ability to insert nucleotides opposite some DNA lesions, pol theta is proficient at extension of unpaired termini. These results show the potential of pol theta to act as an extender after incorporation of nucleotides by other DNA polymerases, and aid in understanding the role of pol theta in somatic mutagenesis and genome instability.

Figure 1. Pol θ efficiently extends mismatched primer termini and weakly from an A opposite the first T of a CPD

A) Substrate used for the assay. A 5’-³²P-labeled 17-mer was annealed to 30-mer DNA. The primer terminus is denoted by X and the template base opposite the primer terminus by Y. B) Extension from primers having a mismatch. Pol θ (168 fmol) was incubated in pol θ buffer for the indicated times with the following substrates, A:T matched (lanes 2–6), A:G mismatch (lanes 8–12), A:C mismatch (lanes 14–18) and T:T mismatch (lanes 20–24). Extension from an A opposite the first T of a CPD was also tested (lanes 24–30). Control experiments without pol θ are shown in lane 1, 7, 13, 19, and 25. Reaction products were separated by electrophoresis on a 20% polyacrylamide DNA sequencing gel and an autoradiograph is shown. C) Quantification of the extension reactions shown in B). Extended products were quantified with a Fuji PhosphoImager and Image Analyzer software. A: G mismatch (open circle), A:C mismatch (open triangle), T:T mismatch (open square) and A:T match (closed circle). Rates of primer-terminus utilization (from data for the first 4 min of incubation) are shown in the inset, with the ratio calculated relative to the matched terminus.

Figure 2. Discrimination against extension of mismatched termini by exo-free Klenow fragment of E. coli pol I

A) Extension from primers having a mismatch. Exo-free Klenow fragment of E. coli pol I (9 x 10⁻⁴ Unit) was incubated in pol I buffer with the same substrates as in Fig. 1: A:T match (lanes 2–6), A:G mismatch (lanes 8–12), A:C mismatch (lanes 14–18) and T:T mismatch (lanes 20–24). Control reaction mixtures without DNA polymerase are in lanes 1, 7, 13, and 19. B) Quantification of the extension reactions. A:G mismatch (open circle), A:C mismatch (open triangle), T:T mismatch (open square) and A:T match (closed circle). Rates of primer-terminus utilization (from data for the first 4 min of incubation) are shown in the inset, with the ratio calculated relative to the matched terminus.

Figure 3. Low discrimination by pol θ for mismatch extension compared to exo-free pol I

Extension reactions from matched or mismatched primers (same as used in Figs. 1 and 2) were tested under identical conditions at pH 8.0 (see Materials and Methods). (A). POLQ (168 fmol) was incubated with A:T matched (lanes 2–4), A:G mismatched (lanes 6–8) or A:C mismatched (lanes 10–12) primer-termini. Exo-free pol I Kf (9 x 10⁻⁴ Unit) was incubated with the same substrates, A:T matched (lanes 14–16), A:G mismatch (lanes 16–20) and A:C mismatch (lanes 22–24). Control experiments without enzymes are shown in lane 1, 5, 9, 13, 17, and 21. Products from part A were quantified for (B), pol θ and (C), exo-free pol I Kf. A: G mismatch (open circle), A:C mismatch (open triangle) and A:T matched (closed circle).

Figure 4. Pol θ extends from an A opposite the second T of a (6–4) photoproduct

(A) A 17-mer (left, lanes 1–12) or an 18-mer (right, lanes 13–24) of the DNA sequence shown was labeled with ³²P at the 5’ end (denoted by the asterisk) and annealed to template 30-mer DNA containing either a (6–4)PP at a TT sequence, or normal TT bases at the same sequence (undamaged). Extension from an A opposite a T of an undamaged template (lanes 2–6) or the first T of a (6–4)PP (lanes 8–12); extension from an A opposite the T of an undamaged template (lanes 14–18) or the second T of a (6–4)PP (lanes 20–24). A time course assay was performed and control experiments without pol θ are shown in lanes 1, 7, 13, and 19. (B) exo-free Klenow fragment of E. coli pol I does not extend from an A opposite a (6–4)PP or a CPD. Extension was tested with 9 x 10⁻³ Unit (x 1) and 9 x 10⁻² Unit (x 10) of enzyme at 37°C for 10 min. Extension reactions from an A opposite the first T of a CPD (lanes 2 and 3), opposite the first T of a (6–4)PP (lanes 5 and 6) and opposite the second T of a (6–4)PP (lanes 8 and 9) were examined. Reaction mixtures with no enzyme are shown in lanes 1, 4 and 7.

Figure 5. Pol θ catalyzes translesion synthesis past a (6–4) photoproduct together with pol ι but not pol η

(A) Substrate used for the assay. 5’-³²P-labeled 16-mer was annealed to 30-mer DNA containing a (6–4)PP, denoted by the bridged TT. (B) The indicated amounts of pol θ were incubated with 630 fmol of pol ι (lanes 3–6) or 63 fmol of pol ι (lanes 9–12) for 10 min at 37°C. 150 fmol of primer-template was used in each reaction mixture. (C) The indicated amounts of pol θ were incubated with 156 fmol of pol η (lanes 2, 4–6) or 15.6 fmol of pol η (lanes 8, 10–12) under the same conditions as in part A.

Figure 6. Extension of pol θ from primer terminus opposite the second T of a (6–4) photoproduct

(A) Substrate used for the assay. A 5′-³²P-labeled 18-mer was annealed to 30-mer DNA containing a (6–4)PP, denoted by the bridged TT. The 3′ terminal base opposite the second T of the (6–4)PP (denoted by N) was varied. (B) Extension from the primer terminus opposite the second T of a 6–4 PP lesion template at 37°C for 10 min in pol θ buffer. Extension from A opposite the second T of a 6–4 PP (lanes 2–6), extension from C (lanes 8–12), extension from a T (lanes 14–18) and extension from a G (lanes 20–24). Control experiments without enzymes are shown in lane 1, 7, 13 and 19.