POLQ (Pol  ), a DNA polymerase and DNA-dependent ATPase in human cells

Abstract

The genomes of eukaryotic cells predict the existence of multiple DNA polymerases, which are proposed to serve specialized roles in DNA replication and repair. We report here the isolation of the full-length human DNA POLQ gene, and an initial characterization of its gene product, DNA polymerase theta. POLQ is of particular interest as it is orthologous to Drosophila Mus308, a gene implicated in cellular resistance to interstrand DNA cross-linking agents. The POLQ cDNA encodes a polypeptide of 2592 amino acids with an ATPase-helicase domain in the N-terminal part of the protein, a central spacer domain, and a DNA polymerase domain in the C-terminal portion. This arrangement is conserved with Mus308. Expression of an mRNA of approximately 8.5 kb was detected in human cell lines. In a survey of human and mouse tissues, expression was highest in testis. Immunoblotting with POLQ antibodies detected a protein of >250 kDa in extracts from HeLa cells. Prominent fragments of approximately 100 kDa suggest that POLQ is readily proteolyzed. Full-length human POLQ was expressed from a baculovirus system. Purified POLQ showed DNA polymerase activity on nicked double-stranded DNA and on a singly primed DNA template. The enzyme activity was resistant to aphidicolin, consistent with its membership of the A family of DNA polymerases, and inhibited by dideoxynucleotides. POLQ further exhibited a single-stranded DNA-dependent ATPase activity.

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

“…Polq mutant mice have an elevated level of spontaneous and radiation-induced micronuclei in reticulocytes [8,9]. POLQ is highly expressed in hematopoietic cells and tissues, including the spleen and germinal centres [9,22,23]. A knockout of POLQ in the chicken DT40 B-cell line shows hypersensitivity to H 2 O 2 , indicating that pol θ is involved in tolerance of damage caused by reactive oxygen species.…”

DNA polymerase θ contributes to the generation of C/G mutations during somatic hypermutation of Ig genes

“…DNA polymerase (Pol ) is a Ϸ300-kDa family A polymerase with a unique structure, having a helicase domain in its N-terminal portion and a polymerase domain in its C terminus (11)(12)(13). Polq, the gene encoding Pol , has a homolog in Drosophila, the mus308 gene (14).…”

“…Mutant flies exhibited hypersensitivity to DNA interstrand crosslinking agents such as cisplatinum but not to alkylating agents such as methyl methanesulfonate (15). Seki et al (13) recently purified human POLQ and examined its catalytic activity. POLQ has extremely low fidelity, making frequent errors on undamaged templates.…”

Mouse models of DNA polymerases

“…In addition to the polymerase activity which is present at the C-terminus, Pol y also possesses an N-terminal helicase domain and single-stranded DNA dependent ATPase activity [Seki et al, 2003]. Pol y can bypass abasic sites by inserting an Adenine opposite the lesion.…”