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.

The Drosophila Mus308 gene is unusual in encoding both a family A DNA polymerase domain and a DNA/ RNA helicase domain. A mus308 mutation was shown to result in increased sensitivity to DNA cross-linking agents, leading to the hypothesis that Mus308 functions in the repair of DNA interstrand cross-links. Recently a mammalian ortholog of Mus308, POLQ, has been identified. We report here the identification, cloning, and characterization of POLN and its gene product, a new mammalian DNA polymerase also related to Mus308. The human cDNA encodes a protein of 900 amino acid residues. The region starting from residue 419 shares 33% identity (48% similarity) with the equivalent region of Escherichia coli DNA polymerase I. POLN is expressed in human cell lines with numerous alternatively spliced transcripts, and a full-length human coding region that comprises 24 exons within 160 kilobases of genomic DNA. Expression analysis by northern blotting and in situ hybridization showed highest expression of full-length POLN in human and mouse testis. POLN localized to the nucleus when expressed as a enhanced green fluorescent protein (GFP)-tagged protein in human fibroblasts. GFP-tagged recombinant POLN had DNA polymerase activity on activated calf thymus DNA and on a singly primed template.A growing number of mammalian DNA polymerases have been identified within recent years, and initial characterization suggests that they have specialized roles in DNA replication or repair. Based on sequence relationships, the enzymes are currently classified into four families designated A, B, X, and Y (1). Founder members of each family are Escherichia coli DNA polymerase (pol) 1 I (family A), mammalian pol ␣ (family B), mammalian pol ␤ (family X), and E. coli pol V (family Y). Nuclear enzymes in families B, X, and Y have received much recent attention, with family Y including enzymes that can bypass DNA template lesions.In Drosophila melanogaster, a family A enzyme is believed to be encoded by the Mus308 gene (2). The COOH-terminal portion of Mus308 is predicted to encode a DNA polymerase, whereas the NH 2 -terminal portion predicts seven characteristic motifs found in DNA and RNA helicases. An apparent human ortholog of Mus308, designated POLQ or DNA pol , is encoded in the human genome (NCBI accession number NM_006596), and a cDNA representing the COOH-terminal part of the gene has been isolated (3). We recently isolated a mammalian DNA helicase gene designated HEL308, which is homologous to the NH 2 -terminal portion of Drosophila Mus308 (4).Mutations in the Mus308 gene lead to marked sensitivity to DNA interstrand cross-linking agents (5). Such interstrand DNA cross-links (ICLs) can be caused by some environmental and chemotherapeutic agents and are potent inhibitors of DNA replication and transcription. In mammalian systems, ICL repair takes place and can be observed in cells, but the mechanisms are not well understood. In E. coli, ICLs can be repaired by the coordinated action of nucleotide excision repair and homologous recombination...