DNA Polymerase Θ: a Cancer Drug Target with Reverse Transcriptase Activity

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) polymerase (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) polymerase 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

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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 polymerase 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 (MRN), confer resistance to etoposide and potentially other DNA-protein crosslinking agents, such as topoisomerase 1 inhibitors. Another recent report found that Polθ-pol unexpectedly exhibits DNA endonuclease activity, which is implicated in end-trimming during MMEJ [39]. Prior studies also discovered 5′-deoxyribose phosphate lyase activity exhibited by the polymerase domain, suggesting a possible function in base excision repair [40].

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lyasePol activityθ has also exhibited been byimplicated the polymerase in the repair domain, of suggestingreplication-associated a possible function DNA breaks, in base whichexcision is supported repair [40]. by its semi-synthetic lethal interaction with ATR [41]. Prior studies demonstratedPolθ has a alsosemi-synthetic been implicated lethal interaction in the repair between of replication-associated Polθ and ATM, which DNA provided breaks, earlywhich evidence is supported in support by its of semi-synthetic Polθ activity in lethal DSB interactionrepair [42]. Lastly, with ATR recent [41 ].studies Prior studiesreveal a demonstratednew function afor semi-synthetic Polθ in suppressing lethal interaction mitotic crossovers between Pol followingθ and ATM, DSBs, which which provided pre- servesearly genome evidence integrity in support by suppressing of Polθ activity the inloss DSB of heterozygosity repair [42]. Lastly, [43,44]. recent Carvajal-Garcia studies reveal eta al. new additionally function for found Polθ thatin suppressingPolθ is synthetic mitotic lethal crossovers with Holliday following junction DSBs, resolvases which pre- (SLX4,serves GEN1), genome which integrity supports by suppressing a model whereby the loss ofPol heterozygosityθ acts on HR intermediates [43,44]. Carvajal-Garcia [43]. Alt- houghet al. additionallymore mechanistic found studies that Pol areθ isneeded synthetic to fully lethal elucidate with Holliday how Pol junctionθ suppresses resolvases mitotic(SLX4, crossovers, GEN1), whichCarvajal-Garcia, supports a J. model et al. suggest whereby that Pol θPolactsθ acts on HRupstream intermediates from Holliday [43]. Al- junctionthough resolvases more mechanistic on HR studiesintermediates are needed where to fullyit can elucidate potentially how perform Polθ suppresses MMEJ [43]. mitotic crossovers, Carvajal-Garcia, J. et al. suggest that Polθ acts upstream from Holliday junction 3.resolvases Polθ Enzymatic on HR intermediatesDomains as Drug where Targets it can potentially perform MMEJ [43].

3. PolAlthoughθ Enzymatic MMEJ Domains is active asduring Drug S-phase, Targets DSB repair is primarily performed by the more accurate HR DSB repair pathway that relies on BRCA1, BRCA2, and associated pro- Although MMEJ is active during S-phase, DSB repair is primarily performed by the teins (i.e., PALB2, RAD51, RAD51 paralogs) (Figure 2A) [45–48]. The DNA nuclease com- more accurate HR DSB repair pathway that relies on BRCA1, BRCA2, and associated proteins plex MRN along with CTBP-interacting protein CtIP is involved in the initial DSB 5′-3′ (i.e., PALB2, RAD51, RAD51 paralogs) (Figure2A) [ 45–48]. The DNA nuclease complex resection process that is required for the generation of 3′ ssDNA overhangs that support MRN along with CTBP-interacting protein CtIP is involved in the initial DSB 50-30 resection HR and MMEJ (Figure 2A) [48,49]. This suggests competition exists between MMEJ and process that is required for the generation of 30 ssDNA overhangs that support HR and MMEJ HR pathways for acting on 3′ ssDNA overhangs [48]. Since MMEJ can serve as a backup (Figure2A) [48,49] . This suggests competition exists between MMEJ and HR pathways for repair pathway in HR-deficient cells (Figure 2B), suppression of Polθ in BRCA deficient acting on 30 ssDNA overhangs [48]. Since MMEJ can serve as a backup repair pathway in cells causes synthetic lethality but has little or no effect in BRCA proficient cells (Figure HR-deficient cells (Figure2B), suppression of Pol θ in BRCA deficient cells causes synthetic 2C) [13,50]. For example, suppression of POLQ gene expression in BRCA1 mutant lethality but has little or no effect in BRCA proficient cells (Figure2C) [ 13,50]. For example, (BRCA1-mut) and BRCA2 mutant (BRCA2-mut) cells resulted in a severe reduction in cell suppression of POLQ gene expression in BRCA1 mutant (BRCA1-mut) and BRCA2 mutant survival(BRCA2-mut) [13,14,51] cells (https://depmap.org/portal resulted in a severe reduction/ccle/ in cell accessed survival on [13 Ma,14y,51 1,]( https://depmap.2021), whereas suppressionorg/portal/ccle/ of POLQ accessed in BRCA on 1 wild-type May 2021), (BRCA-WT) whereas suppression cells had little of POLQ or noin effect BRCA [13,14,51]. wild-type Consistent(BRCA-WT) with cells this, had BRCA-deficient little or no effect cancer [13,14,51 cells]. Consistent were shown with to this, be BRCA-deficientmore dependent cancer on Polcellsθ expression were shown for to their be more survival dependent in the onpresen Polθceexpression of genotoxic for their agents survival and PARPi in the presence[14,23]. Thisof genotoxic synthetic agentslethal relationship and PARPi [ 14between,23]. This Pol syntheticθ and HR lethal was relationshipalso demonstrated between in Pol mouseθ and modelsHR was [14]. also demonstrated in mouse models [14].

ABCHR-proficient cells HR-deficient cells HR-deficient cells DSB DSB DSB -3’ 5’- -3’ 5’- -3’ 5’- -5’ 3’- -5’ 3’- -5’ 3’- MRN-CtIP, 5'-3' resection 5'-3' resection 5'-3' resection ExoI, Dna2, RPA 3’ 5’ 3’ 5’ 3’ 5’ 5’ 3’ 5’ 3’ 5’ 3’ BRCA1/2, RAD51, RAD51 paralogs RPA RPA X RPA Homologous Polθ-dependent Homologous Polθ-dependent Homologous Polθ-dependent recombination MMEJ recombination MMEJ recombination MMEJ (Cell survival) (Cell survival) (Cell death)

Figure 2. Model of Polθ dependency in HR-deficient cells. (A) DSBs are predominantly repaired in S/G2 cell cycle phases by Figure 2. Model of Polθ dependency in HR-deficient cells. (A) DSBs are predominantly repaired in S/G2 cell cycle phases θ byaccurate accurate HR, HR, which which is promotedis promoted by BRCA1/2,by BRCA1/2, RAD51, RAD51, and and associated associated proteins. proteins. Pol -dependentPolθ-dependent MMEJ MMEJ serves serves as a backup as a 0 backupDSB repair DSB pathwayrepair pathway that acts that on 3actsssDNA on 3′ ssDNA overhangs overhangs and causes and large causes indels. large ( Bindels.) HR-deficient (B) HR-deficient cells rely cells on Pol relyθ-dependent on Polθ- dependentMMEJ for MMEJ DSB repair for DSB and repair cell survival. and cell (C survival.) Inactivation (C) Inactivation or suppression or suppression of Polθ causes of Pol syntheticθ causes lethality synthetic in HR-deficientlethality in HR-deficientcells as a result cells of as insufficient a result of DNAinsufficient repair. DNA repair.

Intriguingly,Intriguingly, the the DNA DNA synthesis synthesis and and ATPase ATPase activities activities of of Pol Polθ-polθ-pol and and Pol Polθ-hel,θ-hel, re- re- spectively,spectively, were were shown shown to to promote promote the the survival survival of of BRCA1-mut BRCA1-mut cells cells [14,52]. [14,52]. For For example, example, respectiverespective inactivation inactivation ofof these these domains domains via via site-specific site-specific CRISPR/Cas9 CRISPR/Cas9 mutagenesis mutagenesis within withinthe endogenous the endogenousPolq gene Polq significantly gene significantly reduced reduced colony colony formation formation of BRCA1-deficient of BRCA1-defi- cells cientversus cells BRCA1-proficient versus BRCA1-proficient cells [52]. Thiscells indicates[52]. This thatindicates pharmacological that pharmacological inhibition ofinhibi- either tionPol θofenzymatic either Polθ domain enzymatic will domain kill BRCA1 will kill deficient BRCA1 cancer deficient cells cancer while cells having while little having or no littleeffect oron no normal effect on cells. normal CRISPR/Cas9 cells. CRISPR knock-out/Cas9 knock-out studies also studies showed also that showed Polθ is that synthetic Polθ

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is synthetic lethal with BRCA2 and other DNA repair factors (i.e., RAD54, Ku70/80, FANCJ) [38,51,53]. Lastly, POLQ gene suppression appears to be additive or synergistic with PARPi and DNA damaging agents in killing HR defective cells [14]. Consistent with this, a recently reported potent and selective Polθ polymerase (Polθ-pol) inhibitor developed by Artios Pharma acts additively with PARPi in HR-deficient cells and selectively Geneskills2021, 12HR-deficient, 1146 cells as a single agent at low micromolar concentrations [54]. Because4 of 13 Polθ confers resistance to IR and topoisomerase inhibitors (i.e., etoposide, camptothecin) [20,22], Polθ inhibitors are also expected to reduce cellular resistance to these widely used cancer therapies thatlethal also with cause BRCA2 andDNA other brea DNAks repair and factors replicative (i.e., RAD54, stress. Ku70/80, Indeed, FANCJ) [ 38Artios,51,53]. Lastly, POLQ gene suppression appears to be additive or synergistic with PARPi and DNA Pharma’s Polθ-pol inhibitordamaging agentssignificantly in killing HR reduced defective cellscellular [14]. Consistentresistance with to this, IR a recently[54]. Pol reportedθ inhibitors are also likelypotent to act and synergisticall selective Polθ polymerasey with platinum-based (Polθ-pol) inhibitor chemotherapy developed by Artios agents Pharma in HR-deficient cells basedacts additively on genetic with studies PARPi in [23]. HR-deficient cells and selectively kills HR-deficient cells as a single agent at low micromolar concentrations [54]. Because Polθ confers resistance A more in-depthto IRunderstanding and topoisomerase of inhibitors the respective (i.e., etoposide, enzymatic camptothecin) functions [20,22], Polof θPolinhibitorsθ-pol and Polθ helicase (Polareθ also-hel) expected in MMEJ to reduce andcellular potentially resistance other to these DNA widely repair used mechanisms cancer therapies will that better inform strategiesalso for cause successfully DNA breaks and developi replicativeng stress. this multi-function Indeed, Artios Pharma’sal protein Polθ-pol as inhibitor a ther- significantly reduced cellular resistance to IR [54]. Polθ inhibitors are also likely to act apeutic target. For example,synergistically although with platinum-based Polθ-pol likely chemotherapy has a single agents in function HR-deficient in cellsMMEJ based (ex- on tension of minimallygenetic paired studies ssDNA [23]. overhangs) (Figure 1C), the helicase appears to promote MMEJ dependent Aand more independent in-depth understanding DNA repa of their respective activities. enzymatic Ceccaldi functions et ofal. Pol reportedθ-pol and Polθ helicase (Polθ-hel) in MMEJ and potentially other DNA repair mechanisms will better that Polθ-hel interactsinform with strategies RAD51 for and successfully suppresse developings toxic this RAD51 multi-functional intermediates protein as a therapeuticto confer a survival advantagetarget. in BRCA-deficient For example, although cells Polθ -pol(Figure likely 3A) has a single[14]. functionHowever, in MMEJ it was (extension subse- of quently found that minimallygenetic pairedmutation ssDNA of overhangs) the previously (Figure1C), characterized the helicase appears Pol toθ promote-hel RAD51 MMEJ dependent and independent DNA repair activities. Ceccaldi et al. reported that Polθ-hel binding site had no effectinteracts on with the RAD51 survival and suppressesof BRCA1-deficient toxic RAD51 intermediatescells [52]. Thus, to confer further a survival research is needed to confirmadvantage the in BRCA-deficient functional relevance cells (Figure of3A) reported [ 14]. However, Polθ it-hel:RAD51 was subsequently interac- found tions. We recently dethatmonstrated genetic mutation that ofPol theθ-hel previously exhibits characterized ATP-dependent Polθ-hel RAD51 DNA binding unwinding site had no effect on the survival of BRCA1-deficient cells [52]. Thus, further research is needed activity with 3′–5′ polarity,to confirm similar the functional to RECQ relevance type of SF2 reported helicases Polθ-hel:RAD51 (Figure interactions. 3B) [55]. However, We recently whether this activitydemonstrated contributes that to PolMMEJθ-hel exhibitsor other ATP-dependent DNA repair DNA activities unwinding remains activity unclear. with 30–50 We previously foundpolarity, that Pol similarθ-hel to utilizes RECQ type the SF2 energy helicases of ATP (Figure hydrolysis3B) [ 55]. However, to dissociate whether RPA this activity contributes to MMEJ or other DNA repair activities remains unclear. We previously from ssDNA overhangsfound that that Polenablesθ-hel utilizes ssDNA the energyannealing of ATP and hydrolysis stimulation to dissociate of MMEJ RPA from (Figure ssDNA 3C) [52]. The ability overhangsof RPA:ssDNA that enables complexes ssDNA annealing to suppress and stimulation MMEJ of in MMEJ mammalian (Figure3C) [ 52cells] . The is consistent with a previouslyability of RPA:ssDNA reported complexes role for toRPA suppress in suppressing MMEJ in mammalian a different cells is consistentform of withmi- a previously reported role for RPA in suppressing a different form of microhomology- crohomology-mediatedmediated DSB DSB repair repair in in yeast yeast [ 56[56].].

A Polθ-hel B C Polθ-hel Polθ-hel RAD51 RPA 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ ATP 3'-5' translocation, ATP Polθ-hel 3'-5' translocation ADP + Pi DNA unwinding ADP + Pi Homologous 5’ recombination 3’ 3’ 5’ 5’ 3’ MMEJ

FigureFigure 3.3.Biochemical Biochemical mechanisms mechanisms of Polθ helicase. of Pol (Aθ) Polhelicase.θ-hel was ( reportedA) Polθ to-hel interact was with reported RAD51 and to suppress interact HR with and toxic RAD51:DNA intermediates via its ATPase activity. (B) Polθ-hel was shown to unwind short double-strand DNA RAD51 and suppress HR and toxic RAD51:DNA intermediates via its ATPase activity. (B) Polθ-hel with 30–50 polarity in an ATP hydrolysis-dependent manner. (C) Polθ-hel was shown to dissociate RPA from ssDNA in an wasATP shown hydrolysis-dependent to unwind manner,short double-strand and evidence indicates DNA that with this 3 activity′–5′ polarity stimulates in MMEJ. an ATP hydrolysis-dependent manner. (C) Polθ-hel was shown to dissociate RPA from ssDNA in an ATP hydrolysis-dependent manner, and evidence indicatesMore recently,that this we activity found thatstimulates Polθ-hel MMEJ. strongly contributes to MMEJ in a puriﬁed system by cooperating with Polθ-pol on ssDNA overhangs, which was independent of its ATPase function [25]. For example, although Polθ-pol can perform MMEJ of DNA More recently, substrateswe found with that short Pol 30 ssDNAθ-hel overhangs,strongly itcontributes is deﬁcient in MMEJto MMEJ of substrates in a purified with long system by cooperating30 ssDNA with Pol overhangsθ-pol ason a resultssDNA of its overhangs, prominent snap-back which replicationwas independent activity on ssDNA of its ATPase function [25]. For example, although Polθ-pol can perform MMEJ of DNA substrates with short 3′ ssDNA overhangs, it is deficient in MMEJ of substrates with long 3′ ssDNA overhangs as a result of its prominent snap-back replication activity on ssDNA (Figure 4A) [25]. So-called snap-back replication results from the enzyme’s unique ability to search for microhomology upstream from the 3′ terminus of ssDNA in cis [25,36]. This

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enables(Figure4 A)Pol [θ25-pol]. So-called to form snap-back a stem-loop replication structure results resembling from the enzyme’sa minimally unique base-paired ability primer-templateto search for microhomology that is efficiently upstream extended from by the the 30 terminus enzyme of(Figure ssDNA 4A) in cis[25]. [25 Full-length,36]. This Polθ, onenables the other Polθ-pol hand, to form exhibited a stem-loop minimal structure snap-back resembling replication a minimally activity base-paired on long primer- 3′ ssDNA overhangstemplate that and is instead efficiently primarily extended performed by the enzyme MMEJ (Figure of long4A) ssDNA [ 25]. Full-length substrates Pol withθ, 3′ ter- 0 minalon the microhomology other hand, exhibited (Figure minimal 4B) snap-back[25]. Moreover, replication an ATPase activity onmutant long 3 versionssDNA of full- overhangs and instead primarily performed MMEJ of long ssDNA substrates with 30 length Polθ, and a Polθ-hel-Polθ-pol fusion protein, which includes a short flexible linker terminal microhomology (Figure4B) [ 25]. Moreover, an ATPase mutant version of full- insteadlength Pol ofθ the, and long a Pol centralθ-hel-Pol domain,θ-pol fusion also performed protein, which efficient includes MMEJ a short of flexiblesubstrates linker with long 3instead′ ssDNA of overhangs the long central [25]. Combining domain, also Pol performedθ-pol and efficient Polθ-hel MMEJ as separate of substrates domains with in trans, however,long 30 ssDNA failed overhangs to promote [25 ].MMEJ Combining of long Pol 3′θ ssDNA-pol and overhangs Polθ-hel as and separate instead domains primarily re- sultedin trans, in however,Polθ-pol failedsnap-back to promote replication MMEJ [25]. of longThese 3 0biochemicalssDNA overhangs structure and function instead studies indicateprimarily that resulted close in cooperation Polθ-pol snap-back between replication physically [25 ].linked These Pol biochemicalθ-pol and structure Polθ-hel is re- quiredfunction for studies MMEJ indicate of DNA that with close long cooperation 3′ ssDNA between overhangs, physically and that linked Pol Polθ-helθ-pol ATPase and activ- 0 ityPol θis-hel not is essential required for MMEJMMEJ, of at DNA least with in this long minimal 3 ssDNA purified overhangs, system and thatthat Pol lacksθ-hel RPA and ATPase activity is not essential for MMEJ, at least in this minimal purified system that lacks other auxiliary DNA repair factors. Overall, these studies indicate the Polθ-hel domain RPA and other auxiliary DNA repair factors. Overall, these studies indicate the Polθ-hel bindsdomain ssDNA binds ssDNAupstream upstream from fromPolθ-pol, Polθ-pol, which which effectively effectively bloc blocksks the the polymerase’s polymerase’s promi- nentprominent snap-back snap-back replication replication activity, activity, and and as as a a result, result, enablesenables Pol Polθ-polθ-pol intermolecular intermolecular mi- crohomologymicrohomology search search as as a a major modemode of of action action (Figure (Figure4B). 4B).

Long ssDNA Polθ-hel ABssDNA Polθ-pol Polθ-pol overhangs 5’ 5’ 3’ 3’ 5’ Central dNTPs Interstrand Intrastrand microhomology domain microhomology search PPi search, DNA extension 5’ Microhomology 5’ 5’ dNTPs DNA extension PPi Microhomology 5’ 5’

MMEJ of long ssDNA overhangs FigureFigure 4.4. Full-lengthFull-length Pol Polθ promotesθ promotes MMEJ MMEJ of long of long 30 ssDNA 3′ ssDNA overhangs. overhangs. (A) Pol θ(A-pol) Pol promotesθ-pol promotes snap-backsnap-back replication replication on on ssDNA ssDNA by intramolecular by intramolecul microhomologyar microhomology search, transient search, microhomologytransient microhomol- ogyannealing, annealing, and subsequent and subsequent extension extension of the minimally of the pairedminimally 30 terminal paired end 3′ ofterminal ssDNA. end This of activity ssDNA. This activitysuppresses suppresses MMEJ of longMMEJ ssDNA of long overhangs ssDNA by overhangs Polθ-pol. ( Bby) Full-length Polθ-pol. Pol(B)θ Full-lengthperforms MMEJ Polθ of performs MMEJlong ssDNA. of long Covalent ssDNA. attachment Covalent of attachment Polθ-hel to of Pol Polθ-polθ-hel through to Pol theθ-pol Polθ throughcentral domain the Pol orθ acentral short domain orpeptide a short linker peptide suppresses linker intramolecular suppresses intramolecul microhomologyar microhomology search and snap-back search replication, and snap-back which replication,results which in promoting results in intermolecular promoting intermolecular microhomology searchmicrohomology and subsequent search MMEJ. and subsequent MMEJ.

Taken together,together, multiple multiple lines lines of of evidence evidence demonstrate demonstrate that that both both Pol θPolenzymaticθ enzymatic do- domains contribute to MMEJ and the survival of HR-deficient cells. Consistent with this, mains contribute to MMEJ and the survival of HR-deficient cells. Consistent with this, small-molecule inhibition of either domain causes the preferential killing of BRCA-deficient small-moleculecells [54,57]. For inhibition example, in of addition either domain to the synthetic causes lethalthe preferential effects observed killing for of Artios BRCA-defi- cientPharma’s cells Pol[54,57].θ-pol For inhibitor, example, a recent in addition paper indicates to the synthetic that small-molecule lethal effects inhibition observed of for Ar- tiosPolθ Pharma’s-hel also induces Polθ-pol synthetic inhibitor, lethality a recent in BRCA-deficient paper indicates cells that [57]. small-molecule On the other hand, inhibition ofsimultaneous Polθ-hel also inactivation induces ofsynthetic both domains lethality may in have BR theCA-deficient largest therapeutic cells [57] index. On in the BRCA- other hand, simultaneousdeficient cells and inactivation potentially of other both DNA domains repair defectivemay have cells, the such largest as those therapeutic deficient in index in BRCA-deficientnon-homologous end-joiningcells and potentially (NHEJ). In supportother DNA of this, repair all of defective the reported cells, synthetic such as lethal those defi- cientstudies in on non-homologous Polθ used either CRISPR/Cas9end-joining (NHEJ). or shRNA In to support respectively of this, knock-out all of orthe strongly reported syn- suppress the expression of Polθ, which is equivalent to simultaneous inactivation of both en- thetic lethal studies on Polθ used either CRISPR/Cas9 or shRNA to respectively knock- zymatic domains (https://depmap.org/portal/ccle/ accessed on 1 May 2021) [13,14,51,58]. out or strongly suppress the expression of Polθ, which is equivalent to simultaneous inactivation of both enzymatic domains (https://depmap.org/portal/ccle/ accessed on 1 May 2021) [13,14,51,58]. The development of a proteolysis targeting chimera (PROTAC) [59– 61] degrader of Polθ therefore, represents a plausible therapeutic option since this would fully abolish all of Polθ activities.

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The development of a proteolysis targeting chimera (PROTAC) [59–61] degrader of Polθ therefore, represents a plausible therapeutic option since this would fully abolish all of Polθ activities.

4. Polθ as a Reverse Transcriptase Considering that Polθ definitively functions in MMEJ and TLS pathways and suppressing mitotic crossovers and has been implicated in anti-recombination activity and base excision repair, the question arises whether it exhibits additional DNA repair activities that contribute to cancer cell proliferation. A curious characteristic of Polθ-pol is its deficient proofreading function due to acquired mutations (Figure1A). Pols lacking exonuclease activity typically exhibit low-fidelity DNA synthesis, and in some cases, this enables TLS activity. Interestingly, inactivating the 30–50 exonuclease activity of some related A-family bacterial Pol I enzymes allows these polymerases to reverse transcribe RNA like retroviral reverse transcriptases (RTs), which lack proofreading activity [62,63]. Because Polθ is a highly error-prone enzyme that can utilize various double-strand and ssDNA substrates and is void of exonuclease activity such as retroviral RTs, we examined in recent studies whether Polθ-pol can additionally utilize RNA as a template and thus act as an RT [64]. To determine whether Polθ-pol performs RNA-dependent DNA synthesis activity, we chose a biochemical approach. Our studies utilizing radio-labeled DNA/RNA primer templates found that Polθ-pol exhibits robust RT activity, similar to retroviral RTs (Figure5A) [64] . Notably, Polθ-pol did not require any specific reaction or buffer conditions and performed RNA-dependent DNA synthesis activity on various template constructs and sequences, and its RT activity mimicked retroviral RTs, such as those expressed by Human Immunode- ficiency Virus (HIV), Avian Myeoloblastosis Virus (AMV), and Moloney Murine Leukemia Virus (M-MulV). Most remarkably, our recent report found that Polθ-pol exhibits a higher fidelity and velocity of 20-deoxyribonucleotide incorporation on RNA versus DNA [64]. This suggests the enzyme was selected to be more accurate and efficient on RNA. In contrast to Polθ-pol, all other recombinant human Pols from the A, X, and B families failed to show any RT activity. Y-family Pols κ and η, however, showed minimal RT activity, resulting in the addition of only a few nucleotides on RNA under identical conditions as Polθ-pol and HIV RT, which fully extended DNA/RNA primer-templates. To probe the mechanism by which Polθ-pol uniquely accommodates DNA/RNA hybrids, we utilized X-ray crystallography to solve the ternary structure of Polθ-pol on a DNA/RNA primer-template with incoming 20,30-dideoxyguanosine triphosphate (ddGTP). Remarkably, the 3.2 Å structure of the Polθ-pol:DNA/RNA:ddGTP complex revealed that the thumb domain undergoes a major structural rearrangement to accommodate the DNA/RNA hybrid when compared to the previously solved structures of Polθ-pol binding to a DNA/DNA primer-template (Figure6A–C) [ 26]. For example, 57% of residues within this subdomain, which interacts with the hybrid double-helix region five base-pairs upstream from the 30 primer terminus, undergo conformational changes from helices to loops (Figure6B). This partial refolding of the thumb domain may be needed to provide the necessary interactions with the DNA/RNA hybrid that is wider than B-form DNA/DNA and takes a slightly different orientation in its interaction with the polymerase (Figure6B ). The 12 Å shift observed for residue K2181 highlights the dramatic reconfiguration of the thumb subdomain (Figure6D). In contrast to the two previously solved structures of Polθ-pol:DNA/DNA:ddGTP complexes, which were in the closed configuration, our Polθ- pol:DNA/RNA:ddGTP complex was captured in the open configuration. Thus, the O-helix in the fingers subdomain is rotated outward, and the incoming ddGTP substrate is partially solvent-exposed (Figure6C). The open to closed conformational change upon dNTP binding by A-family Pols on DNA/DNA has been extensively characterized by many laboratories and continues to provide intriguing mechanistic insight into these highly conserved enzymes [65–69]. Another notable shift observed in the Polθ-pol:DNA/RNA:ddGTP complex includes residue E2246 (palm subdomain), which appears to form a specific ribose 20-hydroxyl interaction with the RNA template (Figure6E). Additional multiple hydrogen Genes 2021, 12, x FOR PEER REVIEW 6 of 13

4. Polθ as a Reverse Transcriptase Considering that Polθ definitively functions in MMEJ and TLS pathways and suppressing mitotic crossovers and has been implicated in anti-recombination activity and base excision repair, the question arises whether it exhibits additional DNA repair activities that contribute to cancer cell proliferation. A curious characteristic of Polθ-pol is its deficient proofreading function due to acquired mutations (Figure 1A). Pols lacking exonuclease activity typically exhibit low-fidelity DNA synthesis, and in some cases, this enables TLS activity. Interestingly, inactivating the 3′–5′ exonuclease activity of some related A-family bacterial Pol I enzymes allows these polymerases to reverse transcribe RNA like retroviral reverse transcriptases (RTs), which lack proofreading activity [62,63]. Because Polθ is a highly error-prone enzyme that can utilize various double-strand and ssDNA substrates and is void of exonuclease activity such as retroviral RTs, we examined in recent studies whether Polθ-pol can additionally utilize RNA as a template and thus act as an RT [64]. To determine whether Polθ-pol performs RNA-dependent DNA synthesis activity, we chose a biochemical approach. Our studies utilizing radio-labeled DNA/RNA primer templates found that Polθ-pol exhibits robust RT activity, similar to retroviral RTs (Figure 5A) [64]. Notably, Polθ-pol did not require any specific reaction or buffer conditions and performed RNA-dependent DNA synthesis activity on various template constructs and sequences, and its RT activity mimicked retroviral RTs, such as those ex- Genes 2021, 12, 1146 pressed by Human Immunodeficiency Virus (HIV), Avian Myeoloblastosis7 Virus of 13 (AMV), and Moloney Murine Leukemia Virus (M-MulV). Most remarkably, our recent report found that Polθ-pol exhibits a higher fidelity and velocity of 2′-deoxyribonucleotide in- corporationbonds are on observed RNA versus between DNA the 2 [64].0-hydroxyl This groupsuggests of the the RNA enzyme template was and selected Thr/Gln to be more accurateresidues and of efficient Polθ-pol on (Figure RNA.6F). In Pol contrastθ residue to Y2391 Pol alsoθ-pol, forms all aother hydrogen recombinant bond with thehuman Pols template ribonucleotide that pairs with the incoming ddGTP (Figure6G). Similar hydrogen from bondsthe A, are X, observed and B families in structures failed of retroviral to show RTs, any indicating RT activity. a similar Y-family binding mechanismPols κ and η, however, onshowed RNA [ 70minimal,71]. Overall, RT activity, the major resulting conformational in the changes addition observed of only by a Pol fewθ-pol nucleotides on on RNAthe under DNA/RNA identical relative conditions to prior structures as Pol ofθ the-pol enzyme and capturedHIV RT, on DNA/DNAwhich fully are extended DNA/RNAunprecedented primer-templates. and reveal that Polθ-pol possesses extraordinary plasticity, which likely explains its unusual promiscuity relative to other Pols.

5' end of 3' end of B GFP ORF GFP ORF C A DNA Microhomology Ribonucleotide Polθ-pol RNA RNA MMEJ GFP lesion 5’ -3’ 5’- 5’ 3’ 3’ 5’ -5’ 3’- reporter 3’ 5’ dNTPs x Reverse transcription Cellular 5'-3' resection PPi transfection Polθ RPA 5’ 3’ 5’ Polθ 3’ 5’ 5’ 3’ 5’ RNA-templated 3’ 5’ DNA synapse x DNA repair synthesis RNA-templated 5’ DNA repair synthesis 5’

Polθ-dependent MMEJ (GFP activation) Figure 5. PolFigureθ-pol 5. Pol RNA-dependentθ-pol RNA-dependent DNA DNA synthesis synthesis activities. activities. ((AA)) Pol Polθ-polθ-pol reverse reverse transcribes transcribes RNA, similarRNA, tosimilar retroviral to retroviral reverse transcriptases.reverse transcriptases. (B) Schematic (B) Schematic of an of MMEJ an MMEJ GFP GFP DNA DNA reporter construct construct used used to detect to detect Polθ RNA-templated Polθ RNA-templated DNA DNA θ repair synthesisrepair synthesis in cells. in (C cells.) Model (C) Model of Pol ofθ Pol TLSTLS opposite opposite a a ribonucleotide lesion. lesion. Our studies also confirmed the ability of Polθ to utilize ribonucleotide template bases Toduring probe its DNAthe mechanism repair activities by which in cells. Pol Forθ instance,-pol uniquely we utilized accommodates a newly developed DNA/RNA hybrids,MMEJ we utilized GFP reporter X-ray assay crystallography in which MMEJ to activity solve results the ternary in activation structure of a linear of Pol GFPθ-pol on a θ DNA/RNAexpression primer-template vector [20]. To probe with Pol RNA-dependentincoming 2′ DNA,3′-dideoxyguanosine synthesis activity in cells,triphosphate the GFP reporter was modified to include multiple ribonucleotides adjacent to the mi- (ddGTP).crohomology Remarkably, tract (Figure the 3.25B). Å Here, structure activation of the of thePol GFPθ-pol:DNA/RNA:ddGTP expression vector required complex re- vealedRNA-dependent that the thumb DNA domain repair synthesis undergoes activity a major during MMEJ.structural Our studiesrearrangement clearly showed to accommodate thethat theDNA/RNA inactivation hybrid of Polθ whenvia CRISPR-Cas9 compared genetic to the engineering previously resulted solved in a structures significant of Polθ- pol bindingreduction to in a MMEJ,DNA/DNA which is primer-template consistent with the (Figure ability of 6A–C) Polθ to utilize[26]. For template example, ribonu- 57% of res- cleotides during its DNA repair synthesis activity during MMEJ (Figure5B). Intriguingly, iduesrecent within studies this reveal subdomain, RNA polymerase which II interacts synthesis ofwith RNA the at DSBs, hybrid which double-helix depends on the region five base-pairsMRN complexupstream [72– from74]. Whether the 3′ primer Polθ acts terminus, on these DSB undergo associated conformational RNA substrates changes that from helicesare to involved loops (Figure in the DNA 6B). damage This partial response refolding remains unknown. of the thumb domain may be needed to Being that multiple studies suggest RNA-mediated DNA repair as a potential mechanism of DNA repair in eukaryotic cells [75–77], our findings suggest a plausible function for Polθ in tolerating ribonucleotides during DNA repair. In one scenario, unrepaired ribonucleotides at or near DSB ends can serve as template bases during DNA repair synthesis by Polθ during MMEJ (Figure5B). The possibility also exists that Pol θ utilizes ribonucleotides during TLS (Figure5C). In support of this idea, ribonucleotides are the most frequently occurring lesion in eukaryotic genomes as a result of their misincorporation by replicative Pols [78]. Thus, in the event that genome embedded ribonucleotides are not efficiently repaired, such as in RNASEH2 deficient cells, replication forks are likely to become arrested. This is owing to the fact that replicative Pols are unable to tolerate template ribonucleotides and stall upon encountering these lesions [78,79]. In this scenario, TLS would be a desirable outcome, especially if the ribonucleotide lesions can be replicated accurately. Although future studies will be needed to test Polθ TLS activity opposite genome-embedded ribonucleotides in cells, the enzyme’s ability to easily accommodate A-form DNA/RNA hybrids relative to other Pols reveals its unique and extraordinary structural plasticity. Hence, it will be interesting to determine whether similar structural rearrangements within the Genes 2021, 12, x FOR PEER REVIEW 7 of 13

provide the necessary interactions with the DNA/RNA hybrid that is wider than B-form DNA/DNA and takes a slightly different orientation in its interaction with the polymerase (Figure 6B). The 12 Å shift observed for residue K2181 highlights the dramatic reconfiguration of the thumb subdomain (Figure 6D). In contrast to the two previously solved structures of Polθ-pol:DNA/DNA:ddGTP complexes, which were in the closed configuration, our Polθ-pol:DNA/RNA:ddGTP complex was captured in the open configuration. Thus, the O-helix in the fingers subdomain is rotated outward, and the incoming ddGTP substrate is partially solvent-exposed (Figure 6C). The open to closed conformational change upon dNTP binding by A-family Pols on DNA/DNA has been extensively characterized by many laboratories and continues to provide intriguing mechanistic insight into these highly conserved enzymes [65–69]. Another notable shift observed in the Polθ- pol:DNA/RNA:ddGTP complex includes residue E2246 (palm subdomain), which appears to form a specific ribose 2′-hydroxyl interaction with the RNA template (Figure 6E). Additional multiple hydrogen bonds are observed between the 2′-hydroxyl group of the RNA template and Thr/Gln residues of Polθ-pol (Figure 6F). Polθ residue Y2391 also forms a hydrogen bond with the template ribonucleotide that pairs with the incoming ddGTP (Figure 6G). Similar hydrogen bonds are observed in structures of retroviral RTs, Genes 2021, 12, 1146 indicating a similar binding mechanism on RNA [70,71]. Overall, the major conforma-8 of 13 tional changes observed by Polθ-pol on the DNA/RNA relative to prior structures of the enzyme captured on DNA/DNA are unprecedented and reveal that Polθ-pol possesses enzyme’sextraordinary thumb plasticity, subdomain which occur likely during explains MMEJ its unusual in order promiscuity to accommodate relative minimally to other pairedPols. ssDNA overhangs in an active conﬁguration within the enzyme’s active site.

B A Refolded Thumb Fingers C open O ix -helix el Thumb -h O

ddGTP Palm

Genes 2021, 12, x FOR PEER REVIEW 8 of 13

Exonuclease like DFE C4 Y2391 E2246 K2181 E2246 Y2391

RNA 12 Å N2470

2’OH N2470

C5 K2181 T2239

G RNA T2239 A6

Y2931

T2243

T2243 ddGTP

Figure 6. Structure of the Polθ-pol:DNA/RNA:ddGTP ternary complex. (A) Overlap of the structures of Polθ-

pol:DNA/RNA:ddGTP (green) and Polθ-pol:DNA/DNA:ddGTP (orange, PDB: 4 × 0 q). The fingers and thumb domains Figure 6. Structure of the Polθ-pol:DNA/RNA:ddGTP ternary complex. (A) Overlap of the structures of Polθ- (boxed in black and blue) differ in the two structures. (B) The thumb domain of Polθ-pol in the DNA/DNA has typical pol:DNA/RNA:ddGTP (green) and Polθ-pol:DNA/DNA:ddGTP (orange, PDB: 4 × 0 q). The fingers and thumb domains α-helix folding (orange), but the helices of the thumb of Polθ-pol in complex with DNA/RNA is partially re-folded to (boxed in black and blue) differ in the two structures. (B) The thumb domain of Polθ-pol in the DNA/DNA has typical α- θ helixloops folding (green). (orange), (C) The but O-helix the helices of the of fingers the thumb of Pol of -polPolθ in-pol complex in complex with with DNA/DNA DNA/RNA has is a closedpartially state re-folded (orange), to loops but the (green).fingers (C of) The Polθ O-helix-pol in complexof the fingers with of DNA/RNA Polθ-pol in havecomplex an open with stateDNA/DNA (in green). has a ( Dclosed) The state conformational (orange), but shift the offingers K2181 ofof Pol theθ-pol thumb in complex domain with between DNA/RNA the closed have Pol anθ open-pol:DNA/DNA state (in green). and ( theD) The open conformational Polθ-pol:DNA/RNA shift of K2181 structures. of the ( Ethumb) In the domainPolθ-pol:DNA/RNA between the structure,closed Pol E2246θ-pol:DNA/DNA forms a hydrogen and bond the withopen the Pol 20-OHθ-pol:DNA/RNA of the RNA template. structures. (F) Additional(E) In the hydrogen Polθ- pol:DNA/RNAbond interactions structure, between E2246 20-OH forms groups a hydrogen of the RNA bond template with the and 2′ Pol-OHθ -polof the residues. RNA template. Zoomed-in (F) images Additional of these hydrogen bonding bondinteractions interactions are shownbetween on 2 the′-OH left. groups (G) The of the main-chain RNA template carbonyl and of Pol Y2931θ-pol in Polresidues.θ-pol formsZoomed-in a hydrogen images bond of these with bonding the 20-OH interactionsof the template are shown ribonucleotide on the left. that (G pairs) The withmain-chain the incoming carbonyl ddGTP of Y2931 site inin thePol active.θ-pol forms a hydrogen bond with the 2′- OH of the template ribonucleotide that pairs with the incoming ddGTP site in the active.

Our studies also confirmed the ability of Polθ to utilize ribonucleotide template bases during its DNA repair activities in cells. For instance, we utilized a newly developed MMEJ GFP reporter assay in which MMEJ activity results in activation of a linear GFP expression vector [20]. To probe Polθ RNA-dependent DNA synthesis activity in cells, the GFP reporter was modified to include multiple ribonucleotides adjacent to the microhomology tract (Figure 5B). Here, activation of the GFP expression vector required RNA- dependent DNA repair synthesis activity during MMEJ. Our studies clearly showed that the inactivation of Polθ via CRISPR-Cas9 genetic engineering resulted in a significant reduction in MMEJ, which is consistent with the ability of Polθ to utilize template ribonucleotides during its DNA repair synthesis activity during MMEJ (Figure 5B). Intriguingly, recent studies reveal RNA polymerase II synthesis of RNA at DSBs, which depends on the MRN complex [72–74]. Whether Polθ acts on these DSB associated RNA substrates that are involved in the DNA damage response remains unknown. Being that multiple studies suggest RNA-mediated DNA repair as a potential mechanism of DNA repair in eukaryotic cells [75–77], our findings suggest a plausible function for Polθ in tolerating ribonucleotides during DNA repair. In one scenario, unrepaired ribonucleotides at or near DSB ends can serve as template bases during DNA repair synthesis by Polθ during MMEJ (Figure 5B). The possibility also exists that Polθ utilizes ribonucleotides during TLS (Figure 5C). In support of this idea, ribonucleotides are the most frequently occurring lesion in eukaryotic genomes as a result of their misincorporation by replicative Pols [78]. Thus, in the event that genome embedded ribonucleotides are not efficiently repaired, such as in RNASEH2 deficient cells, replication forks are likely to become arrested. This is owing to the fact that replicative Pols are unable to tolerate template

Genes 2021, 12, 1146 9 of 13

5. Conclusions and Perspectives The continued and growing interest in Polθ biology and drug development is expected to lead to many exciting discoveries regarding the respective activities of Polθ helicase and polymerase, as well as inform the development of improved therapeutics targeting this multi-functional enzyme. The recent report on Artios Pharma’s allosteric Polθ-pol inhibitor class demonstrates for the first time that this specific domain of Polθ is indeed druggable. Another recent study indicates that the helicase domain is also druggable. Here, the authors re-purposed the antibiotic Novabiocin as a Polθ-hel inhibitor that exhibited high micromolar IC50. Further development of potent and selective Polθ-hel inhibitors will be needed to fully assess the potential of this domain as a drug target in HR-deficient cells. As with all early-stage drugs, it remains to be seen whether Polθ-pol or Polθ-hel inhibitors will lead to clinically effective therapeutics. Notably, decades of drug development research targeting HIV RT and other viral polymerases demonstrate the successful development of selective and potent inhibitors against these polymerases, including both competitive nucleoside prodrugs and allosteric non-nucleoside drug inhibitors [80–84]. For example, the prodrug nucleotide analog remdesivir was recently approved for emergency use against Sars-Cov-2 RNA polymerase [85,86]. Clinical grade drug development against human DNA helicases, however, has yet to be achieved. However, considering that the ATP binding pocket within Polθ-hel is well defined and solvent-exposed like kinases [24], this domain will likely be a viable drug target for clinical-grade candidates specifically designed as Polθ-hel inhibitors. A major question that arises from the recent research published by Chandramouly et al. is whether Polθ’s RT activity specifically contributes to particular DNA repair functions, such as cellular tolerance to genome embedded ribonucleotides. Separation of function mutations may be needed to pursue such questions. Considering that Polθ’s RT activity appears to be similar to HIV and other retroviral RTs, a question that arises is whether HIV RT nucleoside inhibitors may be useful for targeting Polθ DNA synthesis activity in cells, and thus, can potentially be re-purposed as anti-cancer agents. Indeed, Polθ and other A-family Pols with a conserved tyrosine residue within the fingers subdomain are known to be inhibited by 20,30,-dideoxyribonucleoside triphosphates (ddNTPs) [27]. Thus, it would be interesting to determine the effects of nucleoside prodrug inhibitors of HIV RT on Polθ cellular activities and the survival of HR-deficient cells. For instance, prodrugs such as zalcitabine and didanosine are converted by nucleoside/nucleotide kinases into their respective active metabolites, ddCTP and ddATP, which inhibit Polθ. The clinical impact of such nucleoside analogs, however, may be limited due to their modest potency and cross-reactivity against mitochondrial Polγ and possibly other Pols. Nevertheless, based on the successful history of competitive nucleotide inhibitors of viral polymerases, the possibility exists that potent and selective nucleotide analog inhibitors of Polθ-pol can be developed in addition to allosteric inhibitors. Regardless of their mechanism of action, the continued development of Polθ-pol and Polθ-hel drug-like inhibitors is expected to generate a lot of excitement for both the translational and basic research communities.

Author Contributions: R.T.P. wrote the review. X.S.C. provided ﬁgures and editorial input. All authors have read and agreed to the published version of the manuscript. Funding: This article was supported by NIH grants 1R01GM130889-01 and 1R01GM137124-01 to R.T.P. and in part by a Tower Cancer Research Foundation grant to X.S.C. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conﬂicts of Interest: R.T.P. and X.S.C. are co-founders of Recombination Therapeutics, LLC. Genes 2021, 12, 1146 10 of 13

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