G C A T T A C G G C A T genes Review DNA Polymerase θ: A Cancer Drug Target with Reverse Transcriptase Activity Xiaojiang S. Chen 1 and Richard T. Pomerantz 2,* 1 Molecular and Computational Biology, USC Dornsife Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089, USA; [email protected] 2 Department of Biochemistry and Molecular Biology, Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107, USA * Correspondence: [email protected] Abstract: The emergence of precision medicine from the development of Poly (ADP-ribose) poly- merase (PARP) inhibitors that preferentially kill cells defective in homologous recombination has sparked wide interest in identifying and characterizing additional DNA repair enzymes that are synthetic lethal with HR factors. DNA polymerase theta (Polθ) is a validated anti-cancer drug target that is synthetic lethal with HR factors and other DNA repair proteins and confers cellular resistance to various genotoxic cancer therapies. Since its initial characterization as a helicase-polymerase fusion protein in 2003, many exciting and unexpected activities of Polθ in microhomology-mediated end-joining (MMEJ) and translesion synthesis (TLS) have been discovered. Here, we provide a short review of Polθ‘s DNA repair activities and its potential as a drug target and highlight a recent report that reveals Polθ as a naturally occurring reverse transcriptase (RT) in mammalian cells. Keywords: DNA polymerase; reverse transcriptase; RNA; reverse transcription; double-strand break Citation: Chen, X.S.; Pomerantz, R.T. repair; translesion synthesis DNA Polymerase θ: A Cancer Drug Target with Reverse Transcriptase Activity. Genes 2021, 12, 1146. https://doi.org/10.3390/ 1. Introduction genes12081146 Mutations in homologous recombination (HR) genes BRCA1 and BRCA2 are strongly predisposed to breast and ovarian cancer [1–6]. Since BRCA deficient cancer cells are im- Academic Editors: Jordi Surralles paired in HR, they are highly susceptible to DNA damage compared to normal cells [4,5]. and Shailja Pathania Drugs that cause DNA damage or inhibit DNA repair, such as Poly (ADP-ribose) poly- merase 1 (PARP1) inhibitors, can therefore cause synthetic lethality in BRCA deficient cells Received: 16 June 2021 while sparing normal cells [4,7–9]. Highly anticipated PARP inhibitors (PARPi), however, Accepted: 20 July 2021 Published: 27 July 2021 lead to drug resistance, which often causes patient mortality [7,10–12]. Thus, it remains important to identify and develop alternative drug targets involved in DNA repair for BRCA deficient cancers that reduce drug resistance and potential side effects. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in Studies performed in 2015 identified the multi-functional DNA repair protein DNA published maps and institutional affil- polymerase θ (Polθ) as a promising drug target in HR-deficient cancers [13,14]. Polθ is iations. upregulated in the majority (70%) of breast tumors and epithelial ovarian cancers [14–18], and its overexpression correlates with HR defects and a poor clinical outcome [14–16,19]. Polθ also confers resistance to ionizing radiation, genotoxic chemotherapy drugs (e.g., topoisomerase inhibitors, cisplatin), and PARPi [14,20–23]. Thus, in addition to promoting the proliferation of HR deficient cells, Polθ’s DNA repair activities, such as microhomology- Copyright: © 2021 by the authors. mediated end-joining (MMEJ) of double-strand breaks (DSBs), contribute to cellular resis- Licensee MDPI, Basel, Switzerland. This article is an open access article tance of a variety of genotoxic anti-cancer agents. distributed under the terms and 2. Overview of Polθ DNA Repair Activities conditions of the Creative Commons Attribution (CC BY) license (https:// Polθ is a large multi-functional protein containing an N-terminal superfamily 2 (SF2) creativecommons.org/licenses/by/ helicase (Polθ-hel) [24], an unstructured central domain, and a C-terminal A-family poly- 4.0/). merase domain (Polθ-pol) that is structurally similar to bacterial Pol I enzymes, such as Genes 2021, 12, 1146. https://doi.org/10.3390/genes12081146 https://www.mdpi.com/journal/genes Genes 2021, 12, x FOR PEER REVIEW 2 of 13 2. Overview of Polθ DNA Repair Activities Polθ is a large multi-functional protein containing an N-terminal superfamily 2 (SF2) helicase (Polθ-hel) [24], an unstructured central domain, and a C-terminal A-family poly- merase domain (Polθ-pol) that is structurally similar to bacterial Pol I enzymes, such as Klenow fragment and Thermus aquaticus (Taq) Pol (Figure 1A) [25–27]. However, in con- Genes 2021, 12, 1146 trast to related Pol I enzymes, Polθ-pol is highly error-prone and is2 promiscuous of 13 in re- gards to its use of nucleic acid and nucleotide substrates [17,28–31]. For example, despite being an A-family polymerase that typically possess relatively high fidelity DNA synthe- Klenow fragment and Thermus aquaticus (Taq) Pol (Figure1A) [ 25–27]. However, in sis, Polcontrastθ-pol to possesses related Pol I enzymes,a deficient Polθ proofreading-pol is highly error-prone domain and due is promiscuous to acquired in mutations, and carriesregards out totranslesion its use of nucleic synthesis acid and nucleotide(TLS) opposite substrates various [17,28–31 ].DNA For example, lesions despite in vitro and in cells, and thereforebeing an A-family is involved polymerase in thatDNA typically damage possess tolerance relatively high (Figure fidelity 1B) DNA [17,31,32]. synthesis, Recent studies Polθ-pol possesses a deficient proofreading domain due to acquired mutations, and carries demonstrateout translesion that synthesis Polθ confers (TLS) opposite resistance various DNAto ultraviole lesions in vitrot (UV)and light-induced in cells, and intrastrand DNAtherefore crosslinks is involved via its in error-prone DNA damage TLS tolerance activity (Figure [33].1B) [The17,31 only,32]. Recentother studiesA-family mammalian Pol knowndemonstrate to thatexhibit Polθ conferserror-prone resistance DNA to ultraviolet synthe (UV)sis light-induced and accommodate intrastrand DNA template lesions is crosslinks via its error-prone TLS activity [33]. The only other A-family mammalian Pol Polνknown, which to exhibitalso lacks error-prone proofreading DNA synthesis activity and accommodate [34]. The templatemajority lesions of TLS is Pol Polsν, belong to the Y-familywhich of also Pols lacks such proofreading as Pol activityκ, Pol [η34, ].and The Pol majorityι, which of TLS possess Pols belong more to the solvent-exposed Y-family active sites ofand Pols are such also as Pol deficientκ, Polη, and in Pol proofreadingι, which possess [35]. more solvent-exposed active sites and are also deficient in proofreading [35]. (Polθ-hel) (Polθ-pol) Aa SF2 helicase domain Central domain A-family Pol domain 1 NT DEAH ~868 ~1,818 2,590 aa ATP binding,Helicase C RAD51 RAD51 Thumb Palm Fingers Palm hydrolysis binding binding Inactive exonuclease domain BCTLS MMEJ DNA lesion DSB 5’ 3’ -3’ 5’- 3’ x 5’ -5’ 3’- RPA MRN-CtIP, Polθ ExoI, Dna2 5'-3' resection Polθ PARP1, RPA 5’ 3’ 5’ 3’ x 5’ 5’ 3’ DNA extension θ HR Pol RPA 5’ 5’ Polθ DNA extension 5’ 5’ FEN1, Ligase 1,3 Figure 1. OverviewFigure 1. Overview of Polθ of Polstructureθ structure and and functi functionon in DNAin DNA repair. repair. (A) Schematic (A) Schematic of full-length of Pol θfull-length.(B) Polθ performs Polθ TLS. (B) Polθ performs θ TLS enablingenabling cellular cellular tolerance tolerance to to DNA DNA damaging damaging agents, agents, such as such ultraviolet as ultraviolet light. (C) Pol light.promotes (C) Pol MMEJ,θ promotes which results MMEJ, in which results the error-prone repair of DSBs and resistance to ionizing radiation and topoisomerase inhibitors. in the error-prone repair of DSBs and resistance to ionizing radiation and topoisomerase inhibitors. Polθ additionally facilitates DSB repair via MMEJ—also referred to as alternative end- joiningPolθ (alt-EJ)additionally and polymerase facilitates theta mediated DSB end-joiningrepair via (TMEJ) MMEJ—also(Figure1C) [13 ,referred21,25,36–38 ]to. as alternative For example, Polθ-pol specifically facilitates MMEJ of DNA with 30 overhangs containing short end-joiningtracts (2–6 bp)(alt-EJ) of microhomology and polymerasein vitro and theta is essential mediated for MMEJ inend-joining cells [13,21,25 ,36(TMEJ),38]. (Figure 1C) [13,21,25,36–38].Recent studies additionally For example, demonstrate Polθ that-pol Pol specificallyθ dependent MMEJ facilitates promotes MMEJ the repair of of DNA 50 with 3′ over- hangsDNA-protein containing crosslinks, short whichtracts can (2–6 form bp) as a of result microhomology of etoposide-induced in covalent vitro and trapping is essential of for MMEJ topoisomerase 2 onto DNA [20]. These studies explain how Polθ and collaborating MMEJ fac- in cellstors, such[13,21,25,36,38]. as Mre11-Rad50-Nbs1 Recent (MRN), studies confer resistanceadditionally to etoposide demonstrate and potentially that other Polθ dependent MMEJDNA-protein promotes crosslinking the repair agents, of such 5′ DNA-protein as topoisomerase 1crosslinks, inhibitors. Another which recent can report form as a result of etoposide-inducedfound that Polθ-pol covalent unexpectedly trapping exhibits DNA of endonucleasetopoisomerase activity, 2 whichonto isDNA implicated [20]. These studies in end-trimming during MMEJ [39]. Prior studies also discovered 50-deoxyribose phosphate explain how Polθ and collaborating MMEJ factors, such as Mre11-Rad50-Nbs1