Promiscuous DNA synthesis by human DNA polymerase θ

Abstract

The biological role of human DNA polymerase θ (POLQ) is not yet clearly defined, but it has been proposed to participate in several cellular processes based on its translesion synthesis capabilities. POLQ is a low-fidelity polymerase capable of efficient bypass of blocking lesions such as abasic sites and thymine glycols as well as extension of mismatched primer termini. Here, we show that POLQ possesses a DNA polymerase activity that appears to be template independent and allows efficient extension of single-stranded DNA as well as duplex DNA with either protruding or multiply mismatched 3'-OH termini. We hypothesize that this DNA synthesis activity is related to the proposed role for POLQ in the repair or tolerance of double-strand breaks.

Figure 1.

Primer extension assays. (a) Extension of properly paired duplex DNA. P denotes the original primer, F denotes full extension to the end of the template and +1 denotes the non-templated addition of a single nucleotide. Extension of single-stranded DNA with (b) purines at the 3′-end of the oligonucleotide, (c) pyrimidines at the 3′-end, (d) purines at the 3′-end of the oligonucleotide with 1 mM ribonucleotides. (e) Extension of duplex DNA with 1 mM ribonucleotides.

Figure 2.

Control experiments. (a) Deletions of loop 1 (residues 2149–2170; Δins1) and loop 2 (residues 2264–2315; Δins2) were tested with double-stranded (ds) or single-stranded (ss) DNA and compared with the wild type polymerase (WT). The DNA templates were those used in Figure 1a and c, respectively, and all four nucleotides were provided. P indicates the original primer, F indicates extension to the end of the template with the duplex DNA and +1 indicates the single non-templated extension past the end of the template with duplex DNA. Reactions were quenched after 30 min. (b) Primer extension by other polymerases. Duplex DNA (ds) is the sequence in Figure 1a and single-stranded DNA (ss) is the sequence in Figure 1c. All four nucleotides were provided and time points were taken at 30 s and 30 min.

Figure 3.

Primer extension assays with mismatched primer termini. (a) Extension past single, double and triple mismatches. (b) Extension of the single-stranded primers as used in the mismatched primer experiments. The sequences are shown above each respective gel.

Figure 4.

Primer extension assays with duplex DNA with varying length of 3′ single-stranded overhangs. The DNA sequences are shown beneath each set of lanes. For each overhang length, the first set of lanes is the duplex DNA substrate and the second set is the single-stranded extension of the primer used without the 14-mer template. In each case, all four dNTPs were supplied. The approximate numbers of nucleotide incorporations for several of the more prominent bands are shown.

Figure 5.

Primer extension assay with duplex DNA with a 3′ single-stranded overhang containing a homopolymeric run of thymines. (a) Extension of the primer alone. (b) Extension of the primer annealed to the template.

Figure 6.

Primer extension assay with homopolymeric substrates. (a) Extension of polyT, polyA and polyC oligonucleotides. (b) Extension of polyTC; where two dNTPs are shown, these were added in equimolar amounts together in the reaction mix.

Figure 7.

Primer extension assay with a single-stranded oligonucleotide designed to be unable to form any self-complimentary base pairs longer than a single nucleotide. The experiment was conducted at both 25 and 37°C and all four dNTPs were provided.