Detection of POLQ expression. Left: northern blot analysis of human POLQ expression in different human tissues. Right: northern blot analysis of human POLQ expression in different mouse tissues. Blots with POLQ probes were exposed to film for 15 days (human) or 8 days (mouse), while the same blots with the control actin probe were exposed for 30 min. The actin probe detected expression of non-muscle γ- and β-actin as well as the α-actin prominent in skeletal and heart muscle.

Analysis of the 5′ end of the POLQ transcript and the POLQ coding sequence. (A) Diagram of the 5′ end of the cDNA determined by 5′ RACE product analysis. The dark gray rectangle is the helicase conserved region (which starts from exon 2). The 5′-non-coding region is shown in white. The black rectangle denotes the predicted nuclear localization signal (NLS). The first ATG in the POLQ ORF is indicated at position +1. The region in which the 5′ RACE primers annealed is shown by an arrow. The lower part of the diagram shows the transcribed DNA sequence around the 5′ end, as found by our 5′ RACE analysis. The dashed vertical line shows the position at which sequence NM_006596 currently predicts a junction between an exon and intron DNA, but in our analysis all of this sequence is part of the transcript. The starting site of each class of 5′ RACE products is indicated with a horizontal line. The numbers in parentheses give the number of products found in MOLT4 or HeLa S3 RNA. (B) Sequence alignment of the coding region of human POLQ (experimentally determined in this study) and mouse POLQ (). 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 conserved helicase motifs I–VI and the conserved DNA polymerase motifs 1–6 are indicated. The predicted NLS is indicated near the beginning of the protein. A conserved Q residue upstream of helicase motif I is indicated by an asterisk, and the conserved Y residue predicting ddNTP sensitivity is indicated with a caret (∧).

POLQ domain structure, conservation and exon arrangement. (A) In the top line of the diagram, the position of a conserved helicase-like domain near the N-terminus of POLQ is shown with gray shading (black vertical bars show the positions of conserved motifs), and the position of a DNA polymerase domain is shown with black shading (white vertical bars show the positions of conserved motifs). Shaded regions in the middle line of the diagram show regions of the protein that are most highly conserved (see Fig. for further detail). At the bottom, the exon structure of the protein is diagrammed. Exon 16 encodes residues 843–1879 of POLQ. Polyclonal antibodies 1 and 2 were raised against regions indicated by brackets. (B) HeLa S3 extract was analyzed by immunoblotting with polyclonal antibodies raised against the central domain of POLQ (positions indicated in the middle line in A). Lane 1, antibody 1; lane 2, antibody 2.

DNA polymerase activity of POLQ. (A) Purified recombinant POLQ. Silver-stained gel showing molecular weight markers (lane 1) and 30 ng of purified POLQ (lane 2). Proteins were separated by electrophoresis on an SDS 4–15% polyacrylamide gradient gel. Two separate samples of 5 ng of POLQ were loaded onto similar gels, immunoblotted with a 1:1000 dilution of POLQ antibody 1 (lane 3) or antibody 2 (lane 4), and detected with secondary reagents as described in Materials and Methods. (B) The hairpin primer–template for the primer extension assay. The self- complementary oligonucleotide was labeled with ³²P at the 5′ end. (C) The primer–template (16 fmol) was incubated with 1.25 ng of POLQ in the presence of a single dNTP (A, T, G or C, lanes 3–6), two dNTPs (lane 7), three dNTPs (lane 8) or all four dNTPs (lane 9). EDTA was added before incubation for the reaction in lane 1, and dNTPs were omitted in lane 2. The reaction mixture in lane 10 included all four dNTPs and 6 × 10 ^–4 U of Taq DNA polymerase (Promega), incubated for 30 min at 74°C.

Sensitivity of POLQ DNA polymerase to inhibitors. (A) ddNTP sensitivity of POLQ on a poly(dA)–oligo(dT)_2:1 template. The indicated amounts of ddTTP were added to reaction mixtures containing 10 µM dTTP. Activities for POLQ (filled circles) and E.coli Klenow fragment (open circles) are expressed as percentages relative to control reactions in the absence of ddTTP. (B) Aphidicolin resistance of POLQ. The reaction conditions were as described in Materials and Methods except that each dNTP was present at 20 µM. Activities for POLQ (filled circles) and calf thymus Pol δ (open circles) are expressed as percentages relative to control reactions in the absence of aphidicolin.

Domain structure of POLQ family homologs. The domain structure of D.melanogaster (Dm) Mus308 (), human (Hs) POLQ (accession no. AY338826) and mouse (Mm) POLQ (accession no. AY074936) is schematized in the upper portion of the diagram. Gray diamonds indicate helicase-like domains, white rectangles the central non-conserved region, and black ovals the polymerase domain. Dotted lines separate the proteins into regions of highest conservation, with the least conserved region in the center. The percentage of sequence similarity/identity between each pair, as determined by a Clustal alignment and MacBoxShade analysis, is indicated by italic numerals. For example, the region between residues 1 and 1028 of human POLQ is 49% similar and 36% identical to a homologous region of Drosophila Mus308 between residues 1 and 1059. Paralogs showing genes closely related to POLQ encode only the helicase-like domain or the polymerase domain and are schematized in the lower portion of the diagram. References or accession numbers: DmHEL308 (NP_648178), Caenorhabditis elegans (Ce) HEL308 (AAF59587), Ce Mus1 (). Hs and Mm POLN (), Hs and Mm HEL308 ().