Search Results (93)

DNA polymerase eta (PolH) is a specialized translesion synthesis DNA polymerase that enables replication across damaged DNA and contributes to cellular tolerance of genotoxic stress. Although PolH has been implicated in chemoresistance, its role in oncogenic signaling remains poorly understood. Here, we identify an unexpected role of PolH in tumor cell growth via mutant p53. We found that overexpression of PolH increases the level of mutant p53 protein. By contrast, knockdown or knockout of PolH inhibits mutant p53 expression by decreasing the half-life of mutant p53 protein. We also found that knockdown or knockout of PolH leads to increased expression of SKP2. As a key E3 ubiquitin ligase component of the SCF complex, we showed that overexpression of SKP2 decreases whereas knockdown of SKP2 increases mutant p53 expression. Moreover, we showed that knockdown of SKP2 abrogates the ability of PolH to regulate mutant p53 expression. To determine the role of SKP2 in mediating mutant p53 proteasome degradation, we showed that SKP2 interacts with mutant p53(R237H). We also found that wild-type but not F-box-deleted SKP2 poly-ubiquitinates mutant p53(R273H). Importantly, we showed that tumor sphere formation is inhibited by knockdown or knockout of PolH, which is further inhibited by knockdown of mutant p53, supporting a cooperative role for PolH and mutant p53 in tumor cell growth. Collectively, these observations indicate that PolH is a previously unrecognized regulator of mutant p53 via SKP2 and that the PolH–mutant p53 pathway can be explored as a therapeutic strategy for mutant p53-driven cancers. Full article

Translesion synthesis is a major DNA damage bypass pathway in which specialized DNA polymerases, such as pol η and Rev1, are recruited to stalled replication forks where they catalyze nucleotide incorporation opposite DNA damage. Translesion synthesis is regulated by the Rad6-Rad18 complex, which catalyzes PCNA mono-ubiquitylation. Despite its central role in regulating translesion synthesis, its oligomeric state and molecular interactions are poorly understood. Here, we use mass photometry to show that Rad18 co-purifies with Rad6 and forms a series of larger oligomeric complexes. Using yeast two-hybrid studies, we showed that while the full-length Rad18 complex interacts with pol η, its interaction with Rev1 is controlled by auto-inhibition. The release of this auto-inhibition is associated with the dissociation of the Rad6-Rad18 complex, freeing Rad18 monomers and dimers. Because pol η participates in non-mutagenic translesion synthesis, while Rev1 participates in mutagenic translesion synthesis, this auto-inhibition likely allows pol η preferential access to stalled replication forks by delaying Rev1 recruitment. Such an ordered polymerase-recruitment mechanism would reduce the likelihood of mutations. Full article

Temozolomide (TMZ) remains the cornerstone of chemotherapeutic agent for glioblastoma (GBM), yet intrinsic and acquired resistance severely limits its clinical benefit. While O⁶-methylguanine-DNA methyltransferase (MGMT)–mediated repair of TMZ-induced O6-methylguanine (O⁶-meG) lesions has been extensively studied, the DNA mismatch repair (MMR) pathway is increasingly recognized as a key determinant of TMZ cytotoxicity. Canonical MMR, mediated by MutSα (MSH2–MSH6) and MutLα (MLH1–PMS2) complexes, recognizes O⁶-meG: thymine mispairs generated during replication and initiates futile repair cycles that culminate in replication stress, replication fork collapse, and apoptotic signaling; intact canonical MMR is, therefore, required for TMZ-induced cell death. Disruption of canonical MMR, frequently via acquired MSH6 mutations, confers TMZ tolerance and drives hypermutated recurrent GBM. Beyond mismatch correction, MMR proteins perform non-canonical functions in DNA damage signaling, replication stress responses, transcriptional regulation, chromatin dynamics, and immune modulation. These activities may shift the outcome from cytotoxic futile repair toward replication stress adaptation, Translesion synthesis (TLS)-mediated lesion tolerance, immune remodeling, and therapeutic resistance. Notably, partial attenuation or functional diversion of MMR may decouple lesion recognition from cytotoxic signaling, enabling TLS-mediated lesion tolerance without complete loss of MMR activity. This review integrates current insights into canonical and non-canonical MMR functions in GBM, defines their distinct contributions to TMZ sensitivity and resistance, and highlights therapeutic opportunities to exploit MMR-associated dependencies, including synthetic lethal strategies and immunotherapeutic vulnerabilities linked to MMR deficiency-driven hypermutation. Full article

Ubiquitin-specific protease 1 (USP1) is an emerging target for poly(ADP-ribose) polymerase 1 (PARP1) inhibitor-resistant and BRCA1/BRCA2 mutant tumors. USP1 is a deubiquitylating enzyme responsible for the removal of the mono-ubiquitin mark on FANCD2, PARP1, and the replication factor proliferating cell nuclear antigen (PCNA), among other proteins. USP1 facilitates proper PCNA-mediated polymerase switching from error-prone trans-lesion synthesis DNA polymerases to replicative DNA polymerases. Due to the critical role of USP1 in DNA synthesis and DNA repair, and the discovery that USP1 deubiquitylates PARP1, USP1 inhibitors (USP1i) were found to have a synthetic lethal relationship with PARP1 inhibitors (PARPi), suggesting a mechanistic link between poly(ADP-ribose) (PAR) dynamics and USP1-mediated ubiquitin hydrolysis. However, the relationship between USP1 inhibition and inhibitors of poly(ADP-ribose) glycohydrolase (PARGi), the primary enzyme responsible for PAR hydrolysis, has not been resolved. Using cell cytotoxicity, synergy, PCNA-ubiquitin, and PAR analyses, it is demonstrated herein that PARG inhibition, combined with USP1 inhibition, leads to increased levels of mono-ubiquitinated PCNA, decreased PAR accumulation, and synergistic cytotoxicity between ML323, a potent USP1i, and PDD00017273, a model PARGi. Future studies will focus on the mechanism that contributes to USP1/PARG synthetic lethality, the mechanism of cell death, and the impact of USP1 on PAR/ubiquitin dynamics and replication stress signaling. Full article

DNA interstrand cross-links (ICLs) are among the most cytotoxic forms of DNA damage, arising when the two strands of the DNA helix are covalently linked by crosslink-inducing agents such as bifunctional alkylating agents and reactive aldehydes. Several studies have demonstrated that ICLs can also be induced by reactive oxygen and nitrogen species. We previously reported that under oxidative conditions, the major oxidative adenine lesion 7,8-dihydro-8-oxoadenine (oxoA) can efficiently generate a novel class of oxoA-G ICLs, structurally resembling guanine–guanine (G–G) cross-links that can be induced by reactive nitrogen species. To investigate the mutagenic potential of these oxidation-induced ICLs in cells, we employed a SupF-based mutagenesis assay using bacterial cells. A single site-specific oxoA–G ICL was synthesized and incorporated into a plasmid, which was then introduced into an E. coli reporter strain to assess mutation profiles induced by both oxoA and oxoA–G ICLs. Our results show that oxoA–G ICLs cause A-to-C/T and G-to-C transversion mutations at the oxoA-G cross-link site, demonstrating highly promutagenic nature of the lesion in bacterial cells. We propose that the oxoA–G ICL may promote transversion mutations, likely driven by a syn conformer of unhooked oxoA-G ICL repair intermediates during translesion synthesis. Full article

Background: Genomic integrity is crucial to the cellular life cycle, which involves a tightly regulated process where cells progress through specific phases to ensure that fully replicated, undamaged DNA is inherited by daughter cells. Any dysfunction in this process or unrepaired DNA damage leads to cell cycle arrest and programmed cell death. Cancer cells are known to exploit these mechanisms to continue dividing. Usually, DNA damage arrests replication, allowing the DNA Damage Response (DDR) pathway to activate, which repairs the DNA or bypasses the damage to support cell survival and preserve genome integrity. For DNA damage bypass or translesion synthesis (TLS), a group of low-fidelity polymerases perform error-prone DNA synthesis opposite damaged bases, where REV1 functions as the main scaffolding protein. Previously, we reported non-TLS functions of REV1, including its role in triggering DNA damage-dependent specific DNA metabolic processes. Methods and Results: In this study, we demonstrate that REV1 plays a significant role in cell cycle progression and that its loss causes arrest at the G2/M phase in flow cytometry analysis. This unexpected phenotype includes dysregulation of G2/M regulators, such as Cyclin B1 and tubulins, in REV1-deficient cells compared to controls, as quantified by Western blot. Additionally, phosphorylation of histone H3 at serine 28 was significantly reduced in these REV1-deficient cells. These G2/M arrest features were even more pronounced in REV1-deficient cells treated with the tubulin inhibitor colchicine. Conclusions: Overall, this study reveals a previously unrecognized link between REV1 TLS polymerase inhibition and the G2/M cell cycle arrest. Full article

Inosine is a key intermediate in many cellular pathways, and our RT-qPCR data showed that DNA polymerase eta (polη) was upregulated upon the repeated treatment of inosine and inosine monophosphate (IMP) in HCT116 cells, which suggests that polη is actively involved in the incorporation and bypass of inosine in cells. To gain novel insight into mutagenic potential of inosine incorporation into DNA and its implication on cell cycle arrest, we conducted structural, biochemical, and cell biological studies of human polη on the incorporation and bypass of inosine. Our nucleotide insertion assay showed that polη incorporated inosine triphosphate (ITP) opposite dC just 18-fold more efficiently than opposite dT, indicating that ITP incorporation by polη is promutagenic. Our three polη crystal structures showed that ITP formed Watson–Crick base pair with dC and that ITP adopted both syn- and anti-conformations across dT, increasing the promutagenicity. Our flow cytometry data showed that only excessive treatment of inosine and IMP caused S- and G2-phase arrest, suggesting that polη’s lesion bypass activity might resolve the cell cycle arrest. Our results give us novel insights into the role of polη in the mutagenic incorporation and bypass of DNA lesions, which might affect cell cycle arrest. Full article

Bacillus subtilis DinG/XPD-like paralogues, DinG and YpvA, have been implicated in overcoming replication stress. DinG possesses a DEDD exonuclease and DNA helicase domains, whereas YpvA lacks the DEDD exonuclease domain. We report that DinG·Mg²⁺ (hereafter referred to as DinG) degrades linear single-stranded (lss) DNA with 3′→5′ polarity and binds lssDNA with higher affinity than its exonuclease-deficient mutant DinG D10A E12A. DinG’s ssDNA-dependent ATPase activity neither stimulates nor inhibits DNA degradation. When bound to the 3′-end of forked DNA, DinG destabilises and degrades the substrate; however, in the presence of ATP, DinG dissociates before reaching the duplex junction. DinG degrades the RNA strand within RNA–DNA hybrids but does not cleave lssRNA unless complexed with Mn²⁺. DinG removes genomic R-loops, as RnhC and PcrA do. DinG physically interacts with RecA and PolA and functions in the same pathway as translesion synthesis (TLS) DNA polymerases (DNAPs) to respond to both spontaneous and methyl methanesulphonate (MMS)-induced mutagenesis. DinG-mGold forms spontaneous foci at or near replication forks, which become enriched following MMS or rifampicin treatment. We propose that DinG contributes to mitigating replication stress by degrading R-loop barriers and facilitating TLS, potentially via RecA-linked mechanisms. Full article

Ovarian cancer (OvCa) is one of the most life-threatening female malignancies that affects 300,000 women annually worldwide. Impaired mechanisms of DNA repair are the leading cause of mutations underlying the OvCa development. microRNAs are short non-coding RNAs that regulate the expression of genes by binding to their transcripts and inducing mRNA degradation or inhibition of translation. Here, we review the miRNA-mediated dysregulation of genes involved in DNA damage response (DDR) and DNA repair pathways in OvCa. Apparently, miRNAs are capable of targeting the crucial mediators of DDR (e.g., miR-203a-3p targeting ATM (Ataxia Telangiectasia Mutated)), homologous repair (such as BRCA1 targeted by miR-9, miR-1255b, miR-193b, and miR-148b), non-homologous end joining (with RNF8 being regulated by miR-214), nucleotide excision repair (involving DDB2 targeted by miR-328-3p), or translesion DNA synthesis (involving RAD18, participating also in homologous repair and targeted by miR-379-5p). We also discuss miRNAs (such as miR-519a-3p, let-7e, miR-216b), which affect responses to OvCa therapy by targeting PARP1 (Poly(ADP-Ribose) Polymerase-1). Finally, we also discuss why, despite the identification of multiple miRNAs capable of regulating DNA repair genes, as well as those involved in the response to therapy, no miRNA-based drugs have been approved for OvCa treatment in clinics. Full article

Replication timing (RT), the temporal order of DNA replication during S phase, influences regional mutation rates, yet the mechanistic basis for RT-associated mutagenesis remains incompletely defined. To identify drivers of RT-dependent mutation biases, we analyzed whole-genome sequencing data from cells with disruptions in DNA replication/repair genes or exposed to mutagenic compounds. Mutation distributions between early- and late-replicating regions were compared using bootstrapping and statistical modeling. We identified 14 genes that exhibit differential effects in early- or late-replicating regions, encompassing multiple DNA repair pathways, including mismatch repair (MLH1, MSH2, MSH6, PMS1, and PMS2), trans-lesion DNA synthesis (REV1) and double-strand break repair (DCLRE1A and PRKDC), DNA polymerases (POLB, POLE3, and POLE4), and other genes central to genomic instability (PARP1 and TP53). Similar analyses of mutagenic compounds revealed 19 compounds with differential effects on replication timing. These results establish replication timing as a critical modulator of mutagenesis, with distinct DNA repair pathways and exogenous agents exhibiting replication timing-specific effects on genomic instability. Our systematic bioinformatics approach identifies new DNA repair genes and mutagens that exhibit differential activity during the S phase. These findings pave the way for further investigation of factors that contribute to genome instability during cancer transformation. Full article

PrimPol is a human DNA primase and DNA polymerase involved in DNA damage tolerance in both nuclei and mitochondria. PrimPol restarts stalled replication forks by synthesizing DNA primers de novo and also possesses DNA translesion activity (TLS activity). PrimPol efficiently and relatively accurately bypasses several DNA lesions including 8-oxoguanine, thymine glycol and 5-formyluracil. In this work, we showed that PrimPol possesses efficient and accurate TLS activity across 8-oxoadenine, another common DNA lesion caused by oxidative stress. The accuracy of PrimPol on DNA with 8-oxoA was significantly higher compared to DNA containing 8-oxoG. Replacement of Mg²⁺ ions with Mn²⁺ stimulated activity of PrimPol on DNA with 8-oxoA and 8-oxoG as well as undamaged A in a sequence-dependent manner by the lesion skipping (or template scrunching) mechanism. Altogether, our data support the idea that PrimPol possesses efficient TLS activity across a wide range of DNA lesions caused by oxidative stress. Full article

Nucleobase and nucleoside analogs are critical components of antimetabolite chemotherapy treatments used to disrupt DNA replication and induce apoptosis in rapidly proliferating cancer cells. However, the development of resistance to these agents remains a major clinical challenge. This review explores the epigenetic mechanisms that contribute to acquired chemoresistance, focusing on DNA methylation, histone modifications, and non-coding RNAs (ncRNAs). These epigenetic alterations regulate key processes such as DNA repair, drug metabolism, cell transport, and autophagy, enabling cancer cells to survive and resist therapeutic pressure. We highlight how dysregulation of DNA methyltransferases (DNMTs) and histone acetyltransferases (HATs) modulates expression of transporters (e.g., hENT1, ABCB1), DNA repair enzymes (e.g., Polβ, BRCA1/2), and autophagy-related genes (e.g., CSNK2A1, BNIP3). Furthermore, emerging roles for long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) in regulating nucleoside export and DNA damage response pathways underscore their relevance as therapeutic targets. The interplay of these epigenetic modifications drives resistance to agents such as gemcitabine and 5-fluorouracil across multiple tumor types. We also discuss recent progress in therapeutic interventions, including DNMT and HDAC inhibitors, RNA-based therapeutics, and CRISPR-based epigenome editing. Full article

A wide variety of endogenous and exogenous alkylating agents covalently modify DNA to produce N7-alkyl-2′-deoxyguanosine (N7-alkylG) adducts as major DNA lesions. The mutagenic potentials of many N7-alkylG adducts with an intercalatable moiety remain poorly understood. We have discovered that the antiriot agent 2-chloroacetophenone readily reacts with dG to produce N7-acetophenone-dG adducts, implicating the genotoxic properties of 2-chloroacetophenone. 2-Chloroacetophenone, however, has been found to be nonmutagenic in both bacterial and mammalian cells. To gain insights into the nonmutagenic nature of N7-acetophenone-dG, we prepared N7-acetophenone-dG-containing oligonucleotide via 2′-fluorine-mediated transition-state destabilization and conducted kinetic and structural studies of human DNA polymerase eta (polη) incorporating nucleotide opposite 2′-F-N7-acetophenone-dG. The kinetic experiments reveal that the presence of the lesion at the templating position greatly hinders nucleotide incorporation. A crystal structure of polη bound to a nonhydrolyzable dCTP analog opposite 2′-F-N7-acetophenone-dG shows that the templating N7-acetophenone-dG is in a syn conformation, precluding binding of an incoming nucleotide in the catalytic site. These unusual conformations explain the observed inefficient incorporation of nucleotide opposite the lesion. Our studies suggest that certain bulky N7-alkylG lesions adopt a syn conformer and present an intercalatable moiety into the nascent base-pairing site, deterring nucleotide incorporation and thus lowering mutagenicity. Full article

The apurinic/apyrimidinic site (AP site) is a highly mutagenic and cytotoxic DNA lesion. Normally, AP sites are removed from DNA by base excision repair (BER). Methoxyamine (MOX), a BER inhibitor currently under clinical trials as a tumor sensitizer, forms adducts with AP sites (AP-MOX) resistant to the key BER enzyme, AP endonuclease. As AP-MOX remains unrepaired, translesion DNA synthesis is expected to be the main mechanism of cellular response to this lesion. However, the mutagenic potential of AP-MOX is still unclear. Here, we compare the blocking and mutagenic properties of AP-MOX and the natural AP site for major eukaryotic DNA polymerases involved in translesion synthesis: DNA polymerases η, ι, ζ, Rev1, and primase–polymerase PrimPol. The miscoding properties of both abasic lesions remained mostly the same for each studied enzyme. In contrast, the blocking properties of AP-MOX compared to the AP site were DNA polymerase specific. Pol η and PrimPol bypassed both lesions with the same efficiency. The bypass of AP-MOX by Pol ι was 15-fold lower than that of the AP site. On the contrary, Rev1 bypassed AP-MOX 5-fold better than the AP site. Together, our data suggest that Rev1 is best suited to support synthesis across AP-MOX in human cells. Full article

Survivin is known for its dual biological role in apoptosis inhibition and mitotic progression. In addition to its being part of the chromosomal passenger complex (CPC), recent findings suggest additional roles for Survivin in the DNA damage response, further contributing to therapy resistance. In this study, we investigated the role of Survivin and the CPC proteins in the cellular response to irradiation with a focus on DNA replication processes. As is known, ionizing radiation leads to an increased expression of Survivin and its accumulation in nuclear foci, which we now know to be specifically localized to centromeric heterochromatin. The depletion of Survivin and Aurora B increases the DNA damage marker γH2AX, indicative of an impaired repair capacity. The presence of Survivin and the CPC in nuclear foci that we already identified during the S phase co-localize with the proliferating cell nuclear antigen (PCNA), further implying a potential role during replication. Indeed, Survivin knockdown reduced replication fork speed as assessed via DNA fiber assays. Mechanistically, we identified a PIP-box motif in INCENP mediating the interaction with PCNA to assist in managing damage-induced replication stress. Survivin depletion forces cells to undergo unphysiological genome replication via mitotic DNA synthesis (MiDAS), resulting in chromosome breaks. Finally, we revealed that Aurora B kinase liberates Pol η by phosphorylating polymerase delta-interacting protein 2 (POLDIP2) to resume the replication of damaged sites via translesion synthesis. In this study, we assigned a direct function to the CPC in the transition from stalled replication forks to translesion synthesis, further emphasizing the ubiquitous overexpression of Survivin particularly in tumors. This study, for the first time, assigns a direct function to the chromosomal passenger complex, CPC, including Survivin, Aurora B kinase, Borealin, and INCENP, in the transition from stalled replication forks (involving PCNA binding) to translesion synthesis (liberating Pol η by phosphorylating POLDIP2), and thus in maintaining genomic integrity. Full article