Exploiting DNA Damage Tolerance for Precision Oncology

Abstract

Unresolved DNA lesions trigger replication stress, forcing cancer cells to hijack DNA damage tolerance (DDT) networks, specifically translesion synthesis (TLS) and template switching, to sustain replication. While DDT prevents lethal fork collapse, error-prone TLS drives mutagenesis, tumor evolution, and chemoresistance/radioresistance. PCNA post-translational modifications dynamically govern pathway selection. Cancer cells exploit this plasticity, creating actionable vulnerabilities like post-replicative ssDNA gaps. Emerging inhibitors targeting TLS polymerases, upstream regulators like USP1, and critical protein-protein interactions offer unprecedented opportunities for precision oncology. By integrating DDT inhibition with biomarkers like homologous recombination deficiency and tumor mutational burden, we can drive synthetic lethality, sensitize tumors to genotoxic agents, suppress treatment-induced mutagenesis, and potentially enhance responses to immunotherapy.

The Dual Nature of DDT in Cancer

Genomic integrity is continually challenged by genotoxic stress [1]. When lesions evade canonical DNA damage response (DDR) pathways (see Glossary) and persist into S phase, cells must deploy DNA damage tolerance (DDT) [2]. DDT operates as a highly coordinated network of core strategies: translesion synthesis (TLS), template switching (TS), and replication fork remodeling that allow replication to proceed across damaged templates, preventing lethal fork collapse (Figure 1a) [3,4].

Figure 1. The DNA damage tolerance ecosystem at stalled replication forks in cancer.

(a) Replication stress arising from chemotherapy, ultraviolet (UV) exposure, oncogenic signaling, and metabolic stress generates stalled replication forks and RPA-coated ssDNA. PCNA post-translational modification contributes to pathway choice: mono-ubiquitination by RAD6–RAD18 promotes error-prone translesion synthesis (TLS), whereas poly-ubiquitination involving HLTF and SHPRH favors error-free template switching (TS). PCNA SUMOylation by PIAS1/PIAS4 contributes to fork regulation and restraint of inappropriate recombination. Fork remodeling and homologous recombination-mediated restart, transcription–replication conflicts and R-loop resolution, and chromatin and epigenetic regulation further shape the response to replication stress. Solid arrows indicate pathway flow, and dotted arrows indicate regulatory influence.

(b) The balance among DDT pathways influences biological outcomes, ranging from TLS-driven mutagenesis, clonal evolution and therapy resistance, and genome instability to error-free replication and preservation of genome stability.

In contemporary oncology, DDT is increasingly recognized as a double-edged sword [5]. Oncogene-induced replication stress (e.g., MYC amplification) and deficiencies in canonical repair (e.g., BRCA1/2 mutations) force tumors into a state of hyper-dependence on DDT pathways to sustain proliferation [6]. While DDT ensures survival under the acute stress of genotoxic chemotherapy, its error-prone TLS branches drive mutagenesis, clonal evolution, and acquired therapeutic resistance (Figure 1b) [7,8]. Understanding how tumor cells hijack these tolerance networks is unlocking an entirely new frontier of precision therapeutics aimed at exploiting both edges of this double-edged sword: targeting the tumor’s dependence on DDT while limiting the mutagenic processes that drive clonal evolution and therapeutic resistance.

Core DNA Damage Tolerance Pathways: Functional Logic of Lesion Bypass

DDT enables replication to proceed across unrepaired DNA lesions through a limited set of mechanistically distinct pathways that balance speed against fidelity [8].

Translesion Synthesis (TLS)

TLS is the most direct mechanism of lesion bypass, utilizing specialized, low-fidelity DNA polymerases; these include Y-family polymerases (Pol η, Pol ι, Pol κ, and REV1) and the B-family polymerase Pol ζ to replicate across damaged bases (Figure 1a, upper left) [8]. All these enzymes possess flexible active sites accommodating distorted DNA templates, typically operating via a two-step mechanism: an “inserter” polymerase incorporates a nucleotide opposite the lesion, followed by extension mediated by Pol ζ [9,10]. As the fastest DDT pathway, TLS prioritizes rapid replication fork progression at the expense of replication fidelity [11].

Template Switching (TS)

In contrast to TLS, TS provides a slower but largely error-free mechanism for lesion bypass by using the undamaged sister chromatid as a template (Figure 1a, upper right) [12]. This recombination-like mechanism allows the replication machinery to bypass lesions without copying damaged DNA and is frequently coupled to post-replicative single-stranded DNA (ssDNA) gap filling [13,14]. Repriming mechanisms, heavily mediated by PrimPol, contribute to this process by enabling continued fork progression while deferring lesion resolution to post-replicative gap-filling pathways [15,16]. Notably, not all replication-associated ssDNA gaps arise through the same mechanism. Besides the physiological post-replicative gaps generated by PrimPol repriming, ssDNA gaps can also arise at, or adjacent to, stalled replication forks following defective fork protection, processing of base excision repair (BER) intermediates such as abasic (AP) sites, or homologous recombination deficiency (HRD). Although both classes of gaps increase dependence on DDT pathways, they differ mechanistically in their origin and biological consequences.

Replication Fork Remodeling

A critical third layer of DDT stabilizes stalled forks to facilitate eventual restart [17]. Unlike TLS or TS, replication fork remodeling does not directly bypass DNA lesions. Instead, it temporarily delays DNA synthesis to stabilize stalled forks, prioritizing genome integrity over replication speed while creating the conditions required for accurate replication restart (Figure 1a, center right). Mediators such as the translocases SMARCAL1, ZRANB3, and HLTF reorganize the replication fork into a four-way junction (fork reversal) [3,18–20]. Fork protection depends critically on factors like BRCA1 and BRCA2, which stabilize RAD51 filaments to prevent nucleolytic processing of replication-associated ssDNA gaps by MRE11 and DNA2 (Figure 1a, center left) [21,22]. Mechanistically, this stabilization is achieved in part via the BRCA2 C-terminal clamp, which restructures RAD51 dimers to facilitate B-DNA binding and enforce replication fork stability [23]. Furthermore, structural and cellular analyses of the RAD51C-XRCC3 complex showed how cancer-associated mutations in RAD51 paralogs compromise these critical DNA replication and fork protection roles [24].

Recent structural and biochemical studies have uncovered an added RAD51 function during DNA replication. Beyond its canonical role in homologous recombination (HR), RAD51 nucleofilaments preferentially recognize AP-containing DNA generated during BER and endogenous base modification pathways. Assembly of RAD51 nucleofilaments on AP-containing DNA shields these lesions from MRE11-dependent cleavage, thereby limiting replication fork breakage and suppressing the accumulation of toxic ssDNA gaps. Similarly, structural insights reveal that EEPD1 acts as a DNA clamping dimer to physically shield reversed replication forks from nucleolytic attack [25]. This multi-protein fork protection mechanism provides a direct molecular connection between BER intermediates, DDT, HR, and Pol θ-dependent gap filling in HR-deficient cells [26].

The recruitment of BRCA1 to orchestrate fork protection and subsequent end resection is tightly regulated by the local epigenetic landscape (Figure 1a, lower right); specifically, heterochromatin protein HP1β recruits the Polycomb protein RING1A to mediate H2AK119 ubiquitination, creating an epigenetic code that facilitates BRCA1 enrichment at damage sites within transcriptionally active regions [27]. In HRD contexts, unprotected reversed forks are targeted for catastrophic nucleolytic degradation, as a primary mechanism underlying the cytotoxicity of crosslinking agents and PARP inhibitors [20,28,29].

In summary, although DDT, replication fork protection, and canonical DNA repair are tightly interconnected, they serve distinct biological functions. DDT pathways enable replication to proceed despite DNA lesions by bypassing or tolerating damage without immediately removing it, whereas replication fork remodeling and protection stabilize stalled forks to prevent collapse until replication can resume. In contrast, canonical DNA repair pathways, including BER, nucleotide excision repair, HR, and non-homologous end-joining, eliminate DNA lesions or repair DNA breaks to restore genome integrity. Functional crosstalk between these mechanisms is extensive, particularly in HRD tumors, where DDT and fork protection become essential for cell survival.

The Sliding Clamp Switchboard: Orchestrating Pathway Choice

The selection between error-prone and error-free lesion bypass is governed by the post-translational modification of Proliferating Cell Nuclear Antigen (PCNA), which serves as the central switchboard directing DDT pathway choice (Figure 1a) [30]. Rather than functioning as a static scaffold, PCNA acts as a dynamic signaling hub [31,32]. Mono-ubiquitination of PCNA at lysine 164 (K164) by the RAD6-RAD18 complex lowers the barrier for polymerase switching, displacing replicative polymerases in favor of specialized, low-fidelity TLS polymerases (e.g., Pol η, Pol κ, REV1) [33,34] (Figure 1a, left arrow). Conversely, the extension of this mark into K63-linked polyubiquitin chains promotes error-free TS, utilizing the sister chromatid for bypass [35,36] (Figure 1a, right arrow). Beyond ubiquitination, PCNA SUMOylation acts as a vital regulatory counterbalance to suppress inappropriate HR during active DNA replication [37] (Figure 1a, downwards arrow). Mechanistically, SUMOylated PCNA recruits anti-recombinogenic helicases, such as PARI or FBH1 in mammalian systems, that actively dismantle RAD51 nucleoprotein filaments and restrict the extension of recombination intermediates [38]. Importantly, this system features intricate molecular crosstalk: SUMOylated PCNA can serve directly as a substrate for ubiquitin ligases [39]. This SUMO-to-ubiquitin handoff enables coordinated transitions between the active suppression of recombination and the rapid activation of lesion bypass pathways [40]. Crucially for therapeutic targeting, this switchboard is reversible. Deubiquitinating enzymes, particularly the USP1/UAF1 complex, remove ubiquitin from PCNA to terminate TLS once a lesion is bypassed [41–43]. In cancer, dysregulation of these ubiquitination cycles frequently biases the cell toward mutagenic TLS, accelerating genome diversification [44]. Collectively, these dynamic post-translational modifications establish PCNA as the central molecular switchboard that integrates replication stress signals and directs pathway choice between error-prone and error-free DDT mechanisms (Figure 1a,b).

DNA Damage Tolerance in Cancer: Survival, Mutagenesis, and Resistance

In cancer cells, the trade-offs inherent to DDT pathway selection are exploited to sustain proliferation under acute replication stress while promoting genetic diversity (Figure 1b) [6]. Elevated expression of TLS polymerases (Pol η, Pol κ, REV1, Pol ζ) is frequently associated with poor prognosis, increased metastatic potential, and intrinsic therapy resistance [6]. Error-prone TLS polymerases introduce base substitutions and small insertions/deletions during lesion bypass, contributing directly to somatic mutations [45,46]. Distinct mutational signatures attributable to TLS polymerases (e.g., COSMIC signatures linked to prior chemotherapy exposure) have been identified across multiple cancer types [47]. This TLS-driven mutagenesis not only fuels tumor evolution but also generates neoantigens, increasing tumor immunogenicity and potentially enhancing responsiveness to immune checkpoint blockade (ICB) [48]. Furthermore, DDT enables replication across therapy-induced DNA lesions, allowing tumor cells to survive treatments like platinum-based agents and radiation [49].

Polymerase-Specific Chemoresistance and Radioresistance

DDT is a major determinant of acquired therapeutic resistance because specific TLS polymerases directly mediate the bypass of distinct chemotherapy-induced lesions [50,51]. For example, Pol η specifically facilitates the bypass of cisplatin-induced DNA adducts, while Pol κ is critical for tolerating alkylation damage induced by agents such as temozolomide [52,53]. Furthermore, specialized polymerases like Pol η not only bypass discrete DNA lesions but also actively facilitate replication through inherently difficult-to-replicate genomic regions, such as common fragile sites, thereby driving genetic variation and promoting cancer cell tolerance of oncogenic replication stress [54]. Elevated expression of these specific polymerases thus strongly correlates with reduced treatment sensitivity, whereas their targeted depletion profoundly re-sensitizes cancer cells to therapy [55,56].

Similarly, ionizing radiation generates a complex spectrum of replication-blocking DNA lesions, including oxidized bases, AP sites, and strand breaks. While TLS polymerases facilitate continued replication across lesions that impede DNA synthesis, replication fork remodeling stabilizes damaged forks until canonical DNA repair pathways resolve more severe DNA damage, including double-strand breaks. Together, these complementary mechanisms contribute substantially to intrinsic radioresistance (Figure 1b). This distinct polymerase-lesion relationship underscores a critical concept for precision oncology: the choice of DDT inhibitor must be rationally tailored to the specific genotoxic backbone of a patient’s chemotherapy and radiotherapy regimen.

Precision Oncology: Therapeutic Disruption of DDT

As tumors inherently rely on DDT to survive both endogenous replication stress and exogenous chemotherapy, targeting this network presents an unprecedented opportunity to induce synthetic lethality and suppress resistance (Figure 2). Therapeutic strategies targeting DDT can be broadly grouped into three categories:

Figure 2. Therapeutic targeting of DDT exploits synthetic lethality in cancer.

In HR-proficient cells (left), inhibition of DDT slows replication but compensation through homologous recombination enables completion of DNA replication and survival. In contrast, HR-deficient tumor cells (right; e.g., BRCA1/2-mutant) rely on DDT for completion of DNA replication; inhibition of DDT targets (e.g., REV1/REV7, RAD18–PCNA, Pol θ and Pol ζ) results in replication fork collapse, double-strand break accumulation, and cell death. This differential dependency creates a therapeutic window for selective tumor killing. Combination strategies integrating DDT inhibition with DNA-damaging agents or PARP inhibitors further enhance cytotoxicity and limit resistance.

Disrupting Protein-Protein Interactions

TLS relies heavily on the scaffold nature of the REV1-Pol ζ complex [57,58]. Small molecules that induce dimerization of the REV1 C-terminal domain and thereby disrupt REV1-REV7 interaction (e.g., JH-RE-06), effectively collapse the TLS machinery [59–62]. Similarly, compounds such as T2 amino alcohol (T2AA) compete with TLS polymerases for PCNA binding via PIP motifs [63]. This approach offers a profound dual advantage: it drastically sensitizes tumors to platinum-based agents while simultaneously starving the cell of its primary mechanism for therapy-induced mutagenesis [64].

Targeting Upstream Regulators

Focusing upstream of the polymerases, inhibitors of the deubiquitinating enzyme USP1 are rapidly reshaping the clinical landscape [65]. USP1 is the deubiquitinating enzyme responsible for removing monoubiquitin from PCNA, thereby limiting recruitment of TLS polymerases. The clinical translation of USP1 inhibitors highlights the profound potential of targeting upstream DDT regulators. Several first-in-class molecules have recently advanced into human studies, including KSQ-4279 and the AI-designed inhibitor ISM3091. These compounds are currently being evaluated in Phase I clinical trials for patients with advanced solid tumors harboring BRCA1/2 mutations or other HR deficiencies. A critical focus of these trials is to test USP1 inhibitors both as monotherapies and in synergistic combinations with PARP inhibitors like olaparib (Figure 2). As USP1 promotes PARP1 release from chromatin, USP1 inhibition enhances PARP1 trapping on chromatin, thereby maximizing replication fork stress. This dual blockade forces toxic, unrestrained TLS while simultaneously creating conditions that compromise BER, a major repair pathway for damaged bases, providing a powerful mechanistic strategy to re-sensitize treatment-refractory tumors and overcome acquired PARP inhibitor resistance.

Within the upstream regulator landscape, Poly(ADP-ribose) glycohydrolase (PARG) also critically modulates the cellular response to replication stress by enforcing p21 degradation via dePARylation to actively promote cancer progression [66]. Consequently, deployment of selective small-molecule PARG inhibitors presents a promising orthogonal therapeutic strategy to actively induce replication fork stalling and drive cancer cell death [67]. Furthermore, pharmacological inhibition of the RAD6-RAD18 ubiquitin ligase complex, which catalyzes PCNA monoubiquitination and initiates TLS, reduces PCNA ubiquitination and enhances chemotherapy sensitivity [68,69]. Together, these strategies suppress TLS by preventing the upstream signaling events required for polymerase recruitment, thereby enhancing the efficacy of DNA-damaging therapies.

Exploiting Vulnerabilities in DNA

The intersection of DDT inhibition with existing targeted therapies is centered on post-replicative ssDNA gaps. In HR-deficient tumors, checkpoint inhibitors (e.g., ATR or Wee1 inhibitors) promote the accumulation of extensive replication-associated ssDNA gaps by abrogating the replication stress checkpoint and allowing cells to progress through S phase before replication-associated ssDNA gaps can be resolved [70]. Whereas canonical TLS polymerases (Pol η, Pol ι, Pol κ, REV1, and Pol ζ) primarily mediate lesion bypass, Pol θ functions predominantly in post-replicative gap filling and theta-mediated end joining, particularly in HR-deficient tumors, where Pol θ inhibition forces lethal mitotic entry [71,72].

This compelling biological rationale has driven the rapid clinical development of specific Pol θ inhibitors designed to target distinct functional domains of the enzyme. For example, the repurposed antibiotic novobiocin is currently in Phase I trials, functioning as a specific allosteric inhibitor of the Pol θ ATPase/helicase domain to selectively kill tumors with alterations in HR genes, including BRCA1, BRCA2, PALB2, and related HR pathway components [73]. Concurrently, novel small molecules such as ART4215, built upon the preclinical success of ART558, have entered clinical trials designed to act as allosteric inhibitors of the Pol θ polymerase catalytic domain [74]. By actively starving cancer cells of their capacity to resolve extensive replication-associated ssDNA gaps, these Pol θ inhibitors force catastrophic mitotic entry, cementing their role as potent sensitizers to DNA-damaging agents and PARP inhibitors in HR-deficient clinical settings (Figure 2).

Because DDT pathways are also essential for maintaining genome integrity in normal proliferating tissues, therapeutic selectivity is a central challenge for clinical development of DDT inhibitors. Yet, cancer cells operate much closer to the threshold of replication catastrophe than normal cells; therefore, therapeutic selectivity is expected to vary across DDT targets. Components such as Pol θ or specific TLS polymerases can be preferentially exploited in tumors experiencing chronic replication stress or HRD, whereas inhibition of broadly essential replication factors, including PCNA, is likely to require greater therapeutic precision to minimize toxicity to normal tissues. These considerations further underscore the importance of biomarker-guided patient selection to maximize therapeutic efficacy while minimizing toxicity.

Biomarker-Driven Stratification

The successful clinical translation of DDT inhibitors will likely require integrated biomarker frameworks that combine genomic, functional, and dynamic indicators of DDT [75].

HR Deficiency and Replication Stress

Existing HRD scores (loss of heterozygosity, telomeric allelic imbalance) successfully identify tumors reliant on tolerance networks [20,76]. To make these frameworks dynamic, they must be integrated with functional indicators of replication stress, including accumulation of RPA-coated ssDNA (detected as RPA nuclear foci), R-loops (DNA-RNA hybrids detected with the S9.6 antibody) (Figure 1a, lower left), persistent γ-H2AX and 53BP1 foci (immunofluorescent markers of ongoing DNA damage signaling), and activation of ATR/CHK1 signaling (typically monitored by phosphorylation-specific antibodies) [77,78]. Together, these functional readouts serve as indirect biomarkers of replication stress because they identify tumor cells operating at the limits of replication fork stability and therefore highly dependent on DNA damage tolerance pathways to maintain viability.

Dynamic Indicators of DDT Engagement

Beyond static protein-level markers, non-coding RNAs introduce a dynamic layer of predictive biomarkers. MicroRNAs, such as miR-96, actively modulate the expression of key DDT and repair components, including REV1 and RAD51 [79]. Profiling these regulatory RNAs can provide real-time prognostic insight into a tumor’s shifting tolerance capacity, reflecting dynamic changes in pathway engagement during sequential therapies. Likewise, elevated expression of polymerases (REV1, POLH, POLK, POLQ) or enhanced mono-ubiquitination of PCNA at lysine 164 will capture the active engagement of TLS recruitment pathways [79–81].

Functional Biomarkers and Therapeutic Response

The genomic “fossil record” of TLS bypass correlates heavily with tumor mutational burden. As mutagenic bypass generates neoantigens, TLS activity profiles are predictive tools for determining which tumors will likely most benefit from the integration of DDT inhibitors with ICB [7,45,82,83]. Besides genomic biomarkers such as BRCA1/2 alterations and HRD signatures, functional biomarkers that directly measure replication-associated ssDNA gaps or persistent BER intermediates may improve patient stratification. Measurements of replication-associated ssDNA gaps, RAD51 loading, RPA accumulation, or AP-site burden provide dynamic indicators of replication stress and DDT that may complement genomic biomarkers in predicting therapeutic response. Several experimental approaches, including native BrdU staining and S1- or APE1-modified DNA fiber assays, have been developed to quantify these functional states [23]. Together, these complementary assays may enable real-time assessment of DDT activity and improve patient stratification beyond static genomic biomarkers.

Tumor-Dependent Therapeutic Outcome

The therapeutic consequences of DDT inhibition are likely to be context dependent. Tumor-specific factors including HRD, intrinsic replication stress, BER activity, TLS polymerase expression, and checkpoint competence (e.g., TP53 or ATR-CHK1 signaling) may collectively determine whether DDT inhibition predominantly induces cytotoxicity, suppresses adaptive mutagenesis, or enhances tumor immunogenicity. Integrating these biological variables with genomic and functional biomarkers will therefore be enabling for selecting patients most likely to benefit from DDT-targeted therapies.

Future Perspectives: Innate Immunity and the cGAS-STING Axis

Emerging evidence highlights a profound link between DDT and tumor immunogenicity [84]. While error-prone lesion bypass directly fuels the generation of novel neoantigens, the structural consequences of disrupting DDT offer an orthogonal immunological trigger. Unresolved replication stress, exacerbated by DDT inhibition, can generate under-replicated DNA that is mis-segregated during mitosis into micronuclei. Subsequent micronuclear envelope rupture releases DNA into the cytosol, where it activates innate immune signaling. Although cytosolic DNA is normally absent from healthy cells, persistent replication stress and chromosome segregation defects promote its pathological accumulation in cancer cells. Cytosolic DNA generated by replication stress, including through MRE11 exonuclease activity, is sensed by cyclic GMP-AMP synthase (cGAS), which activates the cGAS-STING innate immune sensing mechanism and initiates type I interferon signaling [85]. Recent evidence reinforces this paradigm in pancreatic cancer, where the epigenetic regulator DPY30, which promotes H3K4 methylation at stalled forks, safeguards their stability; consequently, therapeutic or genetic disruption of DPY30 destabilizes these stressed forks, triggering acute cGAS-STING-mediated inflammation that robustly sensitizes otherwise recalcitrant tumors to ICB [86].

These observations raise the compelling implication that the targeted modulation of DDT may synergistically enhance responses to immunotherapy. By simultaneously elevating mutation-driven neoantigen burden and triggering cGAS-STING-mediated inflammatory signaling, DDT-targeted strategies provide a compelling mechanistic rationale for combination with ICB [87,88]. Emerging preclinical evidence suggests that inhibition of DDT pathways may increase tumor immunogenicity and sensitize otherwise poorly inflamed (‘cold’) tumors to ICB. The targeted disruption of the structural adaptor GRB2, which normally stabilizes RAD51 at reversed forks to suppress genomic instability, provides an example of how disruption of a DDT-associated fork protection mechanism can activate innate anti-tumor immunity [89]. However, definitive clinical evidence is still lacking. Thus, the immune-priming effect holds profound clinical implications that merit testing for overcoming intrinsic chemoresistance and radioresistance. As tumors exploit DDT to survive DNA damage, including the complex DNA breaks generated by ionizing radiation, combining DDT inhibitors with radiotherapy may not only dismantle this localized survival mechanism but also massively amplify the release of cytosolic DNA. This synergistic DNA damage can hyper-activate cGAS-STING signaling, profoundly remodeling the immunosuppressive tumor microenvironment (TME) by driving the recruitment of cytotoxic lymphocytes [88]. Consequently, leveraging DDT inhibition alongside radiotherapy could serve as a highly effective primer to convert radioresistant, immunologically “cold” TMEs into inflamed hubs optimized for ICB [86].

Concluding Remarks

Historically viewed as a fundamental biochemical failsafe, DDT is now recognized as a critical, under-exploited vulnerability in modern molecular oncology. By enabling replication across DNA lesions, DDT shields cancer cells from catastrophic accumulation of DNA damage and replication failure while simultaneously driving the mutagenesis that fuels tumor evolution and acquired resistance. Strategies that inhibit DDT therefore have the unprecedented potential to restore the efficacy of DNA-damaging treatments, actively limit mutagenesis, exploit synthetic lethal interactions in HRD backgrounds, and likely enhance responses to immunotherapy. As novel agents targeting TLS polymerases and upstream regulators enter the clinic, successful implementation will depend heavily on moving toward precision strategies guided by robust biomarker frameworks and rational combination therapies (see Outstanding Questions). As our understanding of DDT continues to evolve, translating these mechanistic insights into biomarker-guided therapeutic strategies may transform one of cancer’s oldest survival mechanisms into one of its greatest therapeutic liabilities.

Outstanding Questions.

• How effectively can targeted inhibition of error-prone TLS polymerases suppress the emergence of acquired chemoresistance/radioresistance and delay tumor relapse in longitudinal clinical settings?

• How does TLS-induced mutagenesis actively shape the tumor neoantigen landscape, and can DDT inhibitors be synergistically combined with ICB to convert “cold” tumors to “hot”?

• What are the most robust, non-invasive liquid biopsy biomarkers (e.g., circulating tumor DNA [ctDNA] or circulating tumor cells [CTCs]) to dynamically track real-time tumor dependence on specific DDT pathways during sequential therapies?

• How do cancer-specific metabolic states and the hypoxic tumor microenvironment reshape PCNA post-translational modification networks that dictate pathway choice between mutagenic TLS and error-free TS?

Highlights.

• Cancer cells hijack DNA damage tolerance (DDT) networks and epigenetic codes (e.g., PCNA, HP1β, DPY30) to bypass lesions, avoiding lethal fork collapse while fueling tumor evolution.

• Post-replicative ssDNA gaps are toxic intermediates that create profound synthetic lethal vulnerabilities in HR-deficient tumors treated with PARP inhibitors.

• First-in-class inhibitors targeting DDT polymerases (Pol θ), complexes (REV1-Pol ζ), and regulators (USP1) are advancing toward clinical translation, with several entering Phase I trials to dismantle therapy resistance.

• Error-prone TLS bypass drives therapy-induced mutagenesis and neoantigen generation, establishing a direct molecular link between DDT and tumor immunogenicity.

• DDT inhibition combined with genotoxic therapy dismantles radio/chemoresistance and hyper-activates cGAS-STING, converting “cold” microenvironments into “hot” hubs primed for immunotherapy.

Glossary

Abasic (AP) site

A DNA lesion lacking a nucleobase, commonly generated during base excision repair and capable of stalling replication forks if left unresolved.

Base Excision Repair (BER)

Core DNA repair pathway that corrects non-helix-distorting lesions caused by oxidation, alkylation, or deamination.

cGAS-STING

An innate immune sensing mechanism that detects misplaced cytosolic DNA (often originating from unresolved replication stress and fork collapse) and triggers an inflammatory response, promoting tumor immunogenicity.

DNA Damage Response (DDR)

A highly conserved network of cellular pathways that sense, signal, and physically repair DNA lesions to maintain genomic stability (in contrast to DDT, which bypasses them).

DNA Damage Tolerance (DDT)

A network of mechanisms allowing DNA replication machinery to bypass unresolved lesions without immediately repairing them, prioritizing cell survival over genomic fidelity.

Homologous Recombination (HR)

An error-free DNA repair pathway that utilizes an intact homologous sequence (such as a sister chromatid) as a template to repair double-strand breaks and restart collapsed forks.

Homologous Recombination Deficiency (HRD)

A condition that prevents cells from accurately repairing double-strand DNA breaks as a result of mutations in homologous recombination genes, such as BRCA1 or BRCA2.

Immune Checkpoint Blockade (ICB)

Immunotherapy targeting inhibitory immune checkpoint pathways (e.g., PD-1/PD-L1 or CTLA-4) to enhance anti-tumor immune responses.

Proliferating Cell Nuclear Antigen (PCNA)

A sliding DNA clamp whose dynamic post-translational modifications (ubiquitination, SUMOylation) act as the central molecular switchboard for DDT pathway selection.

Replication Fork Remodeling

Structural reorganization of stalled replication forks (e.g., fork reversal) into four-way junctions to protect nascent DNA from nucleolytic degradation.

Single-stranded DNA (ssDNA) Gaps

Vulnerable, unreplicated stretches of DNA that may arise, for example, behind a progressing replication fork following lesion bypass, or during processing of endogenous DNA lesions (e.g., BER intermediates such as abasic sites); resolving these toxic intermediates is a primary target of DDT inhibitors.

Synthetic Lethality

A therapeutic paradigm where the simultaneous disruption of two genetic pathways (e.g., homologous recombination deficiency and DDT inhibition) results in cell death, while the disruption of either alone remains viable.

Template Switching (TS)

An error-free DDT mechanism that utilizes the undamaged sister chromatid as a template to safely bypass lesions.

TransLesion Synthesis (TLS)

An error-prone DDT pathway utilizing specialized, low-fidelity polymerases (e.g., Pol η, Pol ζ, REV1) to replicate directly across damaged DNA templates.