Role of DNA polymerase θ in tolerance of endogenous and exogenous DNA damage in mouse B cells

Abstract

DNA polymerase θ θ θ θ (Polθ θ θ θ ) is a family A polymerase that contains an intrinsic helicase domain. To investigate the function of Polθ θ θ θ in mammalian cells, we have inactivated its polymerase activity in CH12 mouse B lymphoma cells by targeted deletion of the polymerase core domain that contains the catalytic aspartic acid residue. Compared to parental CH12 cells, mutant cells devoid of Polθ θ θ θ polymerase activity exhibited a slightly reduced growth rate, accompanied by increased spontaneous cell death. In addition, mutant cells showed elevated sensitivity to mitomycin C, cisplatin, etoposide, γ γ γ γ -irradiation and ultraviolet (UV) radiation. Interestingly, mutant cells were more sensitive to the alkylating agent methyl methanesulfonate (MMS) than parental cells. This elevated MMS sensitivity relative to WT cells persisted in the presence of methoxyamine, an inhibitor of the major base excision repair (BER) pathway, suggesting that Polθ θ θ θ is involved in tolerance of MMS through a mechanism that appears to be different from BER. These results reveal an important role for Polθ θ θ θ in preventing spontaneous cell death and in tolerance of not only DNA interstrand cross-links and double strand breaks but also UV adducts and alkylation damage in mammalian lymphocytes.

DNA polymeraseθ up-regulation is associated with poor survival in breast cancer, perturbs DNA replication, and promotes genetic instability

“…Nevertheless, we cannot rule out the possibility that mechanisms other than DNA replication could be involved. Excess POLQ could titrate cofactors that are involved in TLS or DNA repair pathways, such as alternative nonhomologous end joining, ICL repair, or BER, where this polymerase might be involved (19)(20)(21)(22)(23). Shorter DNA replication tracts and a modest sensitivity to alkylating agents in POLQ-overexpressing cells could therefore result from an accumulation of DNA damage due to these defective DNA transactions.…”

“…Because POLQ has been proposed to play a role in BER and translesion synthesis (TLS) (19,21,22,25), we hypothesized that overexpression of POLQ could interfere with the cellular tolerance to DNA damage (including damage generated by reactive oxygen species), thus leading to reduced kinetics of replication fork progression. Therefore, we measured the sensitivity of the POLQ overexpressing MRC5-SV cells to increasing doses of methylmethane-sulfonate (MMS) and N-Nitroso-N-Methylurea (MNU) (Fig.…”

“…The observation that chaos1 mutant mice display enhanced micronuclei in red blood cells further suggests a role of POLQ in maintaining genomic stability in the absence of any exogenous stress. In addition it seems to be involved in tolerance of bulky adducts or in DNA repair pathways such as the base-excision repair (BER), DNA interstrand cross-link (ICL), and DNA break repair pathways (19)(20)(21)(22)(23). To investigate the molecular consequences of POLQ up-regulation, we generated human MRC5-SV cells that stably overexpressed the POLQ protein.…”

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

“…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. CH12 mouse B lymphoma cells with a knockdown of pol θ DNA polymerase activity showed elevated sensitivity to UV irradiation, mitomycin C, cisplatin, etoposide, γ-irradiation and MMS [24]. It is important to explore the biochemical capabilities of the pol θ enzyme in order to fully understand the detailed function of pol θ in maintaining genome stability.…”

Kinetic Investigation of the Inhibitory Effect of Gemcitabine on DNA Polymerization Catalyzed by Human Mitochondrial DNA Polymerase

“…3A) (22). Additionally, DNA polymerase , the only other member of the A-family DNA polymerases yet identified in humans, has been shown to bypass abasic site and thymine glycol DNA lesions but is completely unable to bypass a variety of DNA lesions resulting from exposure to UV light and cisplatin-based chemotherapy drugs (46). However, the ability of pol to bypass dFdCMP or any other nucleoside analog within the context of DNA has yet to be examined.…”