Targeting the DNA Repair Enzyme Polymerase θ in Cancer Therapy
Authors: Anna Schrempf, Jana Slyskova, Joanna I. Loizou
Journal: Trends in cancer (2020)

POLQ is a versatile DNA repair enzyme that is central in TMEJ for the error-prone repair of DNA DSBs. POLQ also functions in other DNA repair pathways including base excision repair, interstrand crosslink repair, and DNA damage tolerance by translesion synthesis.Cancer cells often acquire mutations in DNA repair genes, making them dependent on remaining DNA repair pathways. Dependence on TMEJ is characterized by an increased POLQ expression which is associated with poor patient prognosis.Depletion of POLQ in POLQ-dependent cancers leads to synthetic lethality. This is well described for malignancies deficient in homologous recombination (e.g., due to mutations in BRCA1 or BRCA2). Hence, the use of POLQ inhibitors might be a promising strategy for targeted cancer therapy. Targeted cancer therapies represent a milestone towards personalized treatment as they function via inhibition of cancer-specific alterations. Polymerase θ (POLQ), an error-prone translesion polymerase, also involved in DNA double-strand break (DSB) repair, is often upregulated in cancer. POLQ is synthetic lethal with various DNA repair genes, including known cancer drivers such as BRCA1/2, making it essential in homologous recombination-deficient cancers. Thus, POLQ represents a promising target in cancer therapy and efforts for the development of POLQ inhibitors are actively underway with first clinical trials due to start in 2021. This review summarizes the journey of POLQ from a backup DNA repair enzyme to a promising therapeutic target for cancer treatment. Targeted cancer therapies represent a milestone towards personalized treatment as they function via inhibition of cancer-specific alterations. Polymerase θ (POLQ), an error-prone translesion polymerase, also involved in DNA double-strand break (DSB) repair, is often upregulated in cancer. POLQ is synthetic lethal with various DNA repair genes, including known cancer drivers such as BRCA1/2, making it essential in homologous recombination-deficient cancers. Thus, POLQ represents a promising target in cancer therapy and efforts for the development of POLQ inhibitors are actively underway with first clinical trials due to start in 2021. This review summarizes the journey of POLQ from a backup DNA repair enzyme to a promising therapeutic target for cancer treatment. To increase efficiency and lower the burden of toxic side effects, a major goal of cancer therapy is to progress from a ‘one-drug-fits-all’ to an individualized treatment approach tailored to the tumor-specific molecular features. Two main targeted therapeutic strategies are currently utilized in cancer treatment, both exploiting cancer-specific vulnerabilities. In the first approach, therapeutic suppression of aberrantly upregulated oncogenes alleviates the growth advantage of cancer cells. The second approach is based on the phenomenon that genetic alterations acquired by tumor cells cause their dependency on other compensatory pathways, loss of which leads to synthetic lethality (see Glossary). Therefore, therapeutic inhibition of pathways that are synthetic lethal with a cancer-specific alteration evokes cellular death in tumor cells while leaving normal cells unharmed [1.Dobzhansky T. Genetics of natural populations; recombination and variability in populations of Drosophila pseudoobscura.Genetics. 1946; 31: 269-290Crossref PubMed Google Scholar]. The recent advent of genome-wide genetic interaction studies has demonstrated the extensive number of synthetic lethal interactions in cancer, many of which can potentially be translated to targeted cancer therapies [2.Han K. et al.Synergistic drug combinations for cancer identified in a CRISPR screen for pairwise genetic interactions.Nat. Biotechnol. 2017; 35: 463-474Crossref PubMed Scopus (250) Google Scholar]. Cancer cells frequently acquire mutations in DNA repair genes and respond by rewiring their DNA repair network to utilize compensatory pathways for survival. Dependency on compensatory DNA repair pathways opens room for the development of cancer-specific small molecule inhibitors. A group of successful drugs that use this mode of action are poly(ADP-ribose) polymerase (PARP) inhibitors, approved for the treatment of BRCA-deficient cancers. The essentiality of PARP for cancer cells with loss-of-function mutations in BRCA1/2 is remarkable as such cancer cells are up to 1000 times more sensitive to PARP inhibitors than healthy cells [3.Farmer H. et al.Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy.Nature. 2005; 434: 917-921Crossref PubMed Scopus (4441) Google Scholar,4.Bryant H.E. et al.Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase.[erratum appears in Nature. 2007 May 17;447(7142):346].Nature. 2005; 434: 913-917Crossref PubMed Scopus (3464) Google Scholar]. Although challenges such as the acquisition of drug resistance need to be faced, the clinical success of inhibitory drugs targeting DNA repair enzymes is highly encouraging. In this context, the DNA-repair enzyme polymerase θ (POLQ) has received increasing attention. POLQ is upregulated in numerous cancers and its overexpression is associated with poor prognosis [5.Kawamura K. et al.DNA polymerase θ is preferentially expressed in lymphoid tissues and upregulated in human cancers.Int. J. Cancer. 2004; 109: 9-16Crossref PubMed Scopus (94) Google Scholar, 6.Lemée F. et al.DNA polymerase θ up-regulation is associated with poor survival in breast cancer, perturbs DNA replication, and promotes genetic instability.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 13390-13395Crossref PubMed Scopus (115) Google Scholar, 7.Higgins G.S. et al.Overexpression of POLQ confers a poor prognosis in early breast cancer patients.Oncotarget. 2010; 1: 175-184Crossref PubMed Scopus (71) Google Scholar, 8.Lessa R. et al.Adult height and head and neck cancer: a pooled analysis within the INHANCE Consortium.Head Neck. 2014; 36: 1391Google Scholar, 9.Allera-Moreau C. et al.DNA replication stress response involving PLK1, CDC6, POLQ, RAD51 and CLASPIN upregulation prognoses the outcome of early/mid-stage non-small cell lung cancer patients.Oncogenesis. 2012; 1: 1-10Crossref Scopus (68) Google Scholar]. Moreover, synthetic lethal interactions between POLQ and multiple DNA repair genes, including factors involved in homologous recombination (such as BRCA1/2), have been identified [10.Shima N. et al.The mouse genomic instability mutation chaos1 is an allele of Polq that exhibits genetic interaction with Atm.Mol. Cell. Biol. 2004; 24: 10381-10389Crossref PubMed Scopus (105) Google Scholar, 11.Wang Z. et al.DNA polymerase (POLQ) is important for repair of DNA double-strand breaks caused by fork collapse.J. Biol. Chem. 2019; 294: 3909-3919Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 12.Ceccaldi R. et al.Homologous-recombination-deficient tumours are dependent on Polθ -mediated repair.Nature. 2015; 518: 258-262Crossref PubMed Scopus (445) Google Scholar, 13.Mateos-Gomez P.A. et al.Mammalian polymerase θ promotes alternative NHEJ and suppresses recombination.Nature. 2015; 518: 254-257Crossref PubMed Scopus (378) Google Scholar, 14.Kelso A.A. et al.Distinct roles of RAD52 and POLQ in chromosomal break repair and replication stress response.PLoS Genet. 2019; 15: 1-28Crossref Scopus (36) Google Scholar, 15.Feng W. et al.Genetic determinants of cellular addiction to DNA polymerase theta.Nat. Commun. 2019; (Published online September 19, 2019. https://doi.org/10.1038/s41467-019-12234-1)Crossref Scopus (57) Google Scholar, 16.Mengwasser K.E. et al.Genetic Screens Reveal FEN1 and APEX2 as BRCA2 Synthetic Lethal Targets.Mol. Cell. 2019; 73 (e6): 885-899Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar]. For these reasons, POLQ inhibitors, currently in development in multiple biotech companies and laboratories, represent a promising cancer treatment strategy and are soon to be tested in clinical trials. In this review, we first focus on the unique protein structure that allows POLQ to fulfill its diverse roles. We further discuss conflicting evidence of whether POLQ suppresses or promotes genetic stability, given that it is an intrinsically error-prone DNA synthesis enzyme. Finally, we address why POLQ meets the criteria of a promising target in cancer therapy and summarize the state-of-the art in POLQ inhibitor development. POLQ is involved in the repair of DNA double-strand breaks (DSBs), the most cytotoxic type of DNA lesion. If unrepaired, DSBs can have deleterious consequences including genomic rearrangements and cell death. Therefore, a specialized network consisting of at least three pathways is responsible for their repair (Figure 1A ). Most DSBs are repaired by canonical nonhomologous end joining (c-NHEJ), a pathway that directly religates DNA ends without extensive processing, by introducing small insertions and deletions at break sites [17.Chang H.H.Y. et al.Non-homologous DNA end joining and alternative pathways to double-strand break repair.Nat. Rev. Mol. Cell Biol. 2017; 18: 495-506Crossref PubMed Scopus (706) Google Scholar]. In S and G2 phases of the cell cycle, when a sister chromatid is available, homologous recombination (HR) is favored as the only precise DSB repair pathway [18.Ranjha L. et al.Main steps in DNA double-strand break repair: an introduction to homologous recombination and related processes.Chromosoma. 2018; 127: 187-214Crossref PubMed Scopus (148) Google Scholar]. POLQ is involved in a third pathway [originally named alternative end joining (alt-EJ) or microhomology-mediated end joining (MMEJ)] that was later termed polymerase theta-mediated end joining (TMEJ) due to requirement of POLQ [19.Koole W. et al.A polymerase theta-dependent repair pathway suppresses extensive genomic instability at endogenous G4 DNA sites.Nat. Commun. 2014; 5: 1-10Crossref Scopus (140) Google Scholar]. TMEJ is initiated by PARP1 recruitment to resected DNA-ends [20.Audebert M. et al.Involvement of poly(ADP-ribose) polymerase-1 and XRCC1/DNA ligase III in an alternative route for DNA double-strand breaks rejoining.J. Biol. Chem. 2004; 279: 55117-55126Abstract Full Text Full Text PDF PubMed Scopus (533) Google Scholar, 21.Wang M. et al.PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways.Nucleic Acids Res. 2006; 34: 6170-6182Crossref PubMed Scopus (572) Google Scholar, 22.Truong L.N. et al.Microhomology-mediated end joining and homologous recombination share the initial end resection step to repair DNA double-strand breaks in mammalian cells.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 7720-7725Crossref PubMed Scopus (282) Google Scholar]. Upon activation by phosphorylated CtIP, 3′ overhangs are generated by helicases such as the MRE11–RAD50–NBS1 (MRN) complex. POLQ then binds to long single-stranded DNA (ssDNA) overhangs generated by 5′–3′ resection of DSBs and anneals sequences with 2–6 base pairs of microhomology to use them as primers for DNA synthesis [23.Chan S.H. et al.Dual roles for DNA polymerase theta in alternative end-joining repair of double-strand breaks in Drosophila.PLoS Genet. 2010; 6: 1-16Crossref Scopus (296) Google Scholar, 24.Kent T. et al.Mechanism of microhomology-mediated end-joining promoted by human DNA polymerase θ.Nat. Struct. Mol. Biol. 2015; 22: 230-237Crossref PubMed Scopus (174) Google Scholar, 25.Ahrabi S. et al.A role for human homologous recombination factors in suppressing microhomology-mediated end joining.Nucleic Acids Res. 2016; 44: 5743-5757Crossref PubMed Scopus (54) Google Scholar]. The stabilized DNA ends are then ligated by LIG3–XRCC1 or LIG1 [26.Simsek D. et al.DNA ligase III promotes alternative nonhomologous end-joining during chromosomal translocation formation.PLoS Genet. 2011; 7: 1-11Crossref Scopus (213) Google Scholar, 27.Liang L. et al.Human DNA ligases I and III, but not ligase IV, are required for microhomology-mediated end joining of DNA double-strand breaks.Nucleic Acids Res. 2008; 36: 3297-3310Crossref PubMed Scopus (109) Google Scholar, 28.Lu G. et al.Ligase I and ligase III mediate the DNA double-strand break ligation in alternative end-joining.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: 1256-1260Crossref PubMed Scopus (50) Google Scholar] (Figure 1A). Repair by TMEJ is error prone and introduces characteristic sequence alterations, also called mutational signatures with two characteristic attributes. Firstly, since POLQ uses microhomologies for strand annealing and yet only a minority of DNA ends contain such regions, end resection is necessary to make microhomologies accessible. The end joining of resected DNA at microhomologous sequences may result in characteristic microhomology-flanked deletions [29.Sallmyr A. Tomkinson A.E. Repair of DNA double-strand breaks by mammalian alternative end-joining pathways.J. Biol. Chem. 2018; 293: 10536-10549Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar]. Secondly, POLQ tends to abort template-dependent extension from an annealed microhomologous sequence and reanneal at secondary sequences. This results in short stretches of de novo DNA that resembles the sequence flanking the break, also called templated insertions [30.Schimmel J. et al.Templated Insertions: A smoking gun for polymerase theta-mediated end joining.Trends Genet. 2019; 35: 632-644Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar]. Templated insertions can originate from the opposite strand (in trans) or from the same strand (in cis), when the protruding ssDNA snaps back on itself [24.Kent T. et al.Mechanism of microhomology-mediated end-joining promoted by human DNA polymerase θ.Nat. Struct. Mol. Biol. 2015; 22: 230-237Crossref PubMed Scopus (174) Google Scholar,31.Kent T. et al.Polymerase θ is a robust terminal transferase that oscillates between three different mechanisms during end-joining.Elife. 2016; 5: 1-25Crossref Scopus (47) Google Scholar]. Most interestingly, templated insertions can be utilized to map genome-wide TMEJ activity and by doing so, TMEJ most likely contributes to a variety of loci mutated in human disorders, emphasizing TMEJ’s role in the etiology of human diseases [30.Schimmel J. et al.Templated Insertions: A smoking gun for polymerase theta-mediated end joining.Trends Genet. 2019; 35: 632-644Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar]. DNA DSB repair pathways are tightly regulated. In the G1 phase of the cell cycle, during which a sister chromatid for HR is unavailable, association of the highly abundant Ku-heterodimer and 53BP1 with free DNA ends inhibits end resection, thereby channeling repair towards c-NHEJ [32.Bunting S.F. et al.53BP1 inhibits homologous recombination in brca1-deficient cells by blocking resection of DNA breaks.Cell. 2010; 141: 243-254Abstract Full Text Full Text PDF PubMed Scopus (1121) Google Scholar]. In G2 and S phases of the cell cycle, however, 53BP1 is removed from DNA ends by phosphorylated CtIP in complex with BRCA1 and MRN, thereby shifting the balance to favor HR. Since TMEJ and HR both require resected DNA ends, they directly compete with each other for the same substrate. POLQ appears to displace RAD51, a key HR factor, from ssDNA via a proposed RAD51-binding domain [12.Ceccaldi R. et al.Homologous-recombination-deficient tumours are dependent on Polθ -mediated repair.Nature. 2015; 518: 258-262Crossref PubMed Scopus (445) Google Scholar] and may also counteract RPA, another HR factor [33.Mateos-Gomez P.A. et al.The helicase domain of Polθ counteracts RPA to promote alt-NHEJ.Nat. Struct. Mol. Biol. 2017; 24: 1116-1123Crossref PubMed Scopus (75) Google Scholar]. Furthermore, depletion of HR proteins such as BRCA1, BRCA2, or RPA, increases the TMEJ-specific mutational signature suggesting that TMEJ factors are negatively regulated by HR factors [25.Ahrabi S. et al.A role for human homologous recombination factors in suppressing microhomology-mediated end joining.Nucleic Acids Res. 2016; 44: 5743-5757Crossref PubMed Scopus (54) Google Scholar]. Notably, due to its high mutagenicity, TMEJ has been considered merely a backup DNA repair pathway. However, it is becoming increasingly evident that TMEJ also functions in the presence of other DSB repair pathways and might be the only available pathway for specific types of DNA lesions [22.Truong L.N. et al.Microhomology-mediated end joining and homologous recombination share the initial end resection step to repair DNA double-strand breaks in mammalian cells.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 7720-7725Crossref PubMed Scopus (282) Google Scholar]. Such lesions include collapsed replication forks with sister chromatids containing replication-obstructing lesions (e.g., an interstrand crosslink) rendering them an unsuitable repair template for HR [34.Wyatt D.W. et al.Essential roles for polymerase θ-mediated end joining in the repair of chromosome breaks.Mol. Cell. 2016; 63: 662-673Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar] (Figure 1B). In Caenorhabditis elegans, POLQ has been shown to be indispensable for repair of G4 quadruplex structures, thereby preventing genomic rearrangements at the expense of small deletions [19.Koole W. et al.A polymerase theta-dependent repair pathway suppresses extensive genomic instability at endogenous G4 DNA sites.Nat. Commun. 2014; 5: 1-10Crossref Scopus (140) Google Scholar]. Future research is needed to assess the precise regulation of TMEJ and HR in order to identify the conditions and in particular the types of DNA lesions, that depend on TMEJ activity. Increasing evidence suggests that POLQ is involved in DNA damage tolerance and repair of lesions other than DSBs. POLQ can function as a translesion synthesis polymerase and thus incorporates nucleotides opposite apurinic/apyrimidinic sites, thymine glycols, and thymidine dimers [35.Seki M. et al.POLQ (Pol θ), a DNA polymerase and DNA-dependent ATPase in human cells.Nucleic Acids Res. 2003; 31: 6117-6126Crossref PubMed Scopus (144) Google Scholar, 36.Hogg M. et al.Crystallographic snapshots of a replicative DNA polymerase encountering an abasic site.EMBO J. 2004; 23: 1483-1493Crossref PubMed Scopus (126) Google Scholar, 37.Hogg M. et al.Lesion bypass activity of DNA polymerase θ (POLQ) is an intrinsic property of the pol domain and depends on unique sequence inserts.J. Mol. Biol. 2011; 405: 642-652Crossref PubMed Scopus (60) Google Scholar, 38.Kusumoto R. et al.Translesion synthesis by human DNA polymerase η across thymine glycol lesions.Biochemistry. 2002; 41: 6090-6099Crossref PubMed Scopus (118) Google Scholar, 39.Takata K.I. et al.Human DNA polymerase N (POLN) is a low fidelity enzyme capable of error-free bypass of 5S-thymine glycol.J. Biol. Chem. 2006; 281: 23445-23455Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 40.Yoon J.H. et al.Error-prone replication through UV Lesions by DNA polymerase θ protects against skin cancers.Cell. 2019; 176: 1295-1309.e15Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar]. In addition, POLQ has been shown to be important for replication and the repair of replication-associated lesions [41.Alexander J.L. et al.Multiple mechanisms contribute to double-strand break repair at rereplication forks in Drosophila follicle cells.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: 13809-13814Crossref PubMed Scopus (16) Google Scholar,42.Roerink S.F. et al.Polymerase theta-mediated end joining of replication-associated DNA breaks in C. elegans.Genome Res. 2014; 24: 954-962Crossref PubMed Scopus (94) Google Scholar]. Depletion of POLQ results in decreased replication fork velocity and an increased amount of stalled replication forks upon treatment with hydroxyurea, a chemical used to induce replication fork stalling [12.Ceccaldi R. et al.Homologous-recombination-deficient tumours are dependent on Polθ -mediated repair.Nature. 2015; 518: 258-262Crossref PubMed Scopus (445) Google Scholar]. DNA single-strand breaks that are converted into DSBs upon encountering replication forks might also depend on repair by POLQ [11.Wang Z. et al.DNA polymerase (POLQ) is important for repair of DNA double-strand breaks caused by fork collapse.J. Biol. Chem. 2019; 294: 3909-3919Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar]. Furthermore, while POLQ appears to be essential for the repair of interstrand crosslinks (ICLs) in Drosophila, Arabidopsis, and C. elegans [43.Beagan K. et al.Drosophila DNA polymerase theta utilizes both helicase-like and polymerase domains during microhomology-mediated end joining and interstrand crosslink repair.PLoS Genet. 2017; 13: 1-19Crossref Scopus (28) Google Scholar, 44.Muzzini D.M. et al.Caenorhabditis elegans POLQ-1 and HEL-308 function in two distinct DNA interstrand cross-link repair pathways.DNA Repair (Amst). 2008; 7: 941-950Crossref PubMed Scopus (73) Google Scholar, 45.Inagaki S. et al.Arabidopsis TEBICHI , with helicase and DNA polymerase domains, is required for regulated cell division and differentiation in meristems.18. 2006: 879-892Google Scholar], most studies in mammalian systems demonstrate that POLQ is not required for this type of repair, potentially due to redundancy with other TLS polymerases [10.Shima N. et al.The mouse genomic instability mutation chaos1 is an allele of Polq that exhibits genetic interaction with Atm.Mol. Cell. Biol. 2004; 24: 10381-10389Crossref PubMed Scopus (105) Google Scholar,46.Yousefzadeh M.J. et al.Mechanism of suppression of chromosomal instability by DNA polymerase POLQ.PLoS Genet. 2014; 10e1004654Crossref PubMed Scopus (154) Google Scholar]. A few exceptions have been reported: POLQ knockout (KO) mouse embryonic fibroblasts (MEFs) have been shown to be hypersensitive to mitomycin C, an ICL-inducing agent, and higher levels of micronuclei in response to mitomycin C were observed in POLQ mutant mice [15.Feng W. et al.Genetic determinants of cellular addiction to DNA polymerase theta.Nat. Commun. 2019; (Published online September 19, 2019. https://doi.org/10.1038/s41467-019-12234-1)Crossref Scopus (57) Google Scholar,47.Shima N. et al.Phenotype-based identification of mouse chromosome instability mutants.Genetics. 2003; 163: 1031-1040Crossref PubMed Google Scholar]. Finally, based on the presence of a weak 5′-deoxyribose phosphate lyase activity in its polymerase domain, POLQ was suggested to act in base excision repair (BER) [48.Prasad R. et al.Human DNA polymerase θ possesses 5′-dRP lyase activity and functions in single-nucleotide base excision repair in vitro.Nucleic Acids Res. 2009; 37: 1868-1877Crossref PubMed Scopus (78) Google Scholar], although the extent of its involvement is a matter of debate [49.Yoshimura M. et al.Vertebrate POLQ and POLβ cooperate in base excision repair of oxidative DNA damage.Mol. Cell. 2006; 24: 115-125Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 50.Ukai A. et al.Role of DNA polymerase θ in tolerance of endogenous and exogenous DNA damage in mouse B cells.Genes Cells. 2006; 11: 111-121Crossref PubMed Scopus (26) Google Scholar, 51.Arana M.E. et al.Low-fidelity DNA synthesis by human DNA polymerase theta.Nucleic Acids Res. 2008; 36: 3847-3856Crossref PubMed Scopus (106) Google Scholar, 52.Higgins G.S. et al.A small interfering RNA screen of genes involved in DNA repair identifies tumor-specific radiosensitization by POLQ knockdown.Cancer Res. 2010; 70: 2984-2993Crossref PubMed Scopus (84) Google Scholar]. In conclusion, POLQ is involved in multiple DNA repair pathways but deeper insights into both the variation between model organisms as well as the mechanistic function of POLQ in each pathway are lacking. POLQ encodes an A-family polymerase that contains both an N-terminal conserved superfamily 2 helicase domain and a C-terminal DNA polymerase domain, linked by an unstructured central region (Figure 2). As such, POLQ is the only eukaryotic polymerase known to date that contains a helicase domain. A coordinated interplay between all domains is necessary to allow for execution of POLQ activity [53.Black S.J. et al.Molecular basis of microhomology-mediated end-joining by purified full-length Polθ.Nat. Commun. 2019; 10 (Published online September 27, 2019. https://doi.org/10.1038/s41467-019-12272-9)Crossref Scopus (28) Google Scholar] (Figure 2). The polymerase domain is responsible for DNA synthesis either using its terminal transferase or templated extension activity. Despite its low sequence conservation, the central domain appears to be important for regulating POLQ substrate selection. A mutant version of POLQ lacking its central domain can perform TMEJ on short ssDNA substrates (≤26 nucleotides) whereas full-length POLQ cannot [53.Black S.J. et al.Molecular basis of microhomology-mediated end-joining by purified full-length Polθ.Nat. Commun. 2019; 10 (Published online September 27, 2019. https://doi.org/10.1038/s41467-019-12272-9)Crossref Scopus (28) Google Scholar]. Finally, the helicase domain contains both ATPase and helicase activity as well as proposed RAD51-binding motifs that may displace RAD51 from ssDNA in an ATPase-dependent manner [12.Ceccaldi R. et al.Homologous-recombination-deficient tumours are dependent on Polθ -mediated repair.Nature. 2015; 518: 258-262Crossref PubMed Scopus (445) Google Scholar,35.Seki M. et al.POLQ (Pol θ), a DNA polymerase and DNA-dependent ATPase in human cells.Nucleic Acids Res. 2003; 31: 6117-6126Crossref PubMed Scopus (144) Google Scholar,54.Büttner K. et al.Structural basis for DNA duplex separation by a superfamily-2 helicase.Nat. Struct. Mol. Biol. 2007; 14: 647-652Crossref PubMed Scopus (242) Google Scholar, 55.Richards J.D. et al.Structure of the DNA repair helicase Hel308 reveals DNA binding and autoinhibitory domains.J. Biol. Chem. 2008; 283: 5118-5126Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 56.Newman J.A. et al.Structure of the helicase domain of DNA polymerase theta reveals a possible role in the microhomology-mediated end-joining pathway.Structure. 2015; 23: 2319-2330Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar]. This domain is required for performing TMEJ on longer ssDNA substrates since binding of the polymerase domain alone results in an unproductive snap-back mechanism [53.Black S.J. et al.Molecular basis of microhomology-mediated end-joining by purified full-length Polθ.Nat. Commun. 2019; 10 (Published online September 27, 2019. https://doi.org/10.1038/s41467-019-12272-9)Crossref Scopus (28) Google Scholar]. In summary, POLQ contains a helicase domain capable of competing with HR for resected DNA-ends and a polymerase domain for strand annealing and extension, connected by a flexible central region. Whether POLQ suppresses or promotes genomic instability is a matter of debate. Biochemical studies have shown that POLQ polymerase activity has low fidelity and its involvement in DSB repair frequently culminates in large deletions and templated insertions [29.Sallmyr A. Tomkinson A.E. Repair of DNA double-strand breaks by mammalian alternative end-joining pathways.J. Biol. Chem. 2018; 293: 10536-10549Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar,30.Schimmel J. et al.Templated Insertions: A smoking gun for polymerase theta-mediated end joining.Trends Genet. 2019; 35: 632-644Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar,57.Seki M. et al.High-efficiency bypass of DNA damage by human DNA polymerase Q.EMBO J. 2004; 23: 4484-4494Crossref PubMed Scopus (161) Google Scholar]. Beyond in vitro systems, various studies performed in mouse and human systems have yielded conflicting findings both supporting and opposing its role as a guardian of genomic stability (Table 1). POLQ has been shown to protect genomic stability: its depletion increases DSB formation, exacerbates sensitivity to various genotoxic agents, and destabilizes replication forks [10.Shima N. et al.The mouse genomic instability mutation chaos1 is an allele of Polq that exhibits genetic interaction with Atm.Mol. Cell. Biol. 2004; 24: 10381-10389Crossref PubMed Scopus (105) Google Scholar,12.Ceccaldi R. et al.Homologous-recombination-deficient tumours are dependent on Polθ -mediated repair.Nature. 2015; 518: 258-262Crossref PubMed Scopus (445) Google Scholar,14.Kelso A.A. et al.Distinct roles of RAD52 and POLQ in chromosomal break repair and replication stress response.PLoS Genet. 2019; 15: 1-28Crossref Scopus (36) Google Scholar,46.Yousefzadeh M.J. et al.Mechanism of suppression of chromosomal instability by DNA polymerase POLQ.PLoS Genet. 2014; 10e1004654Crossref PubMed Scopus (154) Google Scholar,52.Higgins G.S. et al.A small interfering RNA screen of genes involved in DNA repair identifies tumor-specific radiosensitization by POLQ knockdown.Cancer Res. 2010; 70: 2984-2993Crossref PubMed Scopus (84) Google Scholar,58.Goff J.P. et al.Lack of DNA polymerase θ (POLQ) radiosensitizes bone marrow stromal cells in vitro and increases reticulocyte micronuclei after total-body irradiation.Radiat. Res. 2009; 172: 165-174Crossref PubMed Scopus (57) Google Scholar]. Other studies, however, have reported that POLQ depletion decreases chromosomal translocations and UV-associated mutations and its overexpression increases DNA damage markers and impairs cell cycle progression [6.Lemée F. et al.DNA polymerase θ up-regulation is associated with poor survival in breast cancer, perturbs DNA replication, and promotes genetic instability.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 13390-13395Crossref PubMed Scopus (115) Google Scholar,13.Mateos-Gomez P.A. et al.Mammalian polymerase θ promotes alternative NHEJ and suppresses recombination.Nature. 2015; 518: 254-257Crossref PubMed Scopus (378) Google Scholar,40.Yoon J.H. et al.Error-prone replication through UV Lesions by DNA polymerase θ protects against skin cancers.Cel