Search Results (39)

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

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

DNA damage tolerance pathways that allow for the completion of replication following fork arrest are critical in maintaining genome stability during cell division. The main DNA damage tolerance pathways include strand switching, replication fork reversal and translesion synthesis (TLS). The TLS pathway is mediated by specialised DNA polymerases that can accommodate altered DNA structures during DNA synthesis, and are important in allowing replication to proceed after fork arrest, preventing fork collapse that can generate more deleterious double-strand breaks in the genome. TLS may occur directly at the fork, or at gaps remaining behind the fork, in the process of post-replication repair. Inactivating mutations in the human POLH gene encoding the Y-family DNA polymerase Pol η causes the skin cancer-prone genetic disease xeroderma pigmentosum variant (XPV). Pol η also contributes to chemoresistance during cancer treatment by bypassing DNA lesions induced by anti-cancer drugs including cisplatin. We review the current understanding of the canonical role of Pol η in translesion synthesis following replication arrest, as well as a number of emerging non-canonical roles of the protein in other aspects of DNA metabolism. Full article

Pierisin is a DNA-targeting ADP-ribosyltransferase found in cabbage white butterfly (Pieris rapae). Pierisin transfers an ADP-ribosyl moiety to the 2-amino group of the guanine residue in DNA, yielding N²-(ADP-ribos-1-yl)-2′-deoxyguanosine (N²-ADPR-dG). Generally, such chemically modified DNA is recognized as DNA damage and elicits cellular responses, including DNA repair pathways. In Escherichia coli and human cells, it has been experimentally demonstrated that N²-ADPR-dG is a substrate of the nucleotide excision repair system. Although DNA repair machineries can remove most lesions, some unrepaired damages frequently lead to mutagenesis through DNA replication. Replication past the damaged DNA template is called translesion DNA synthesis (TLS). In vitro primer extension experiments have shown that eukaryotic DNA polymerase κ is involved in TLS across N²-ADPR-dG. In many cases, TLS is error-prone and thus a mutagenic process. Indeed, the induction of G:C to T:A and G:C to C:G mutations by N²-ADPR-dG in the hypoxanthine phosphoribosyltransferase gene mutation assay with Chinese hamster cells and supF shuttle vector plasmids assay using human fibroblasts has been reported. This review provides a detailed overview of DNA repair, TLS and mutagenesis of N²-ADPR-dG induced by cabbage butterfly pierisin-1. Full article

Translesion synthesis (TLS) is a mechanism of DNA damage tolerance utilized by eukaryotic cells to replicate DNA across lesions that impede the high-fidelity replication machinery. In TLS, a series of specialized DNA polymerases are employed, which recognize specific DNA lesions, insert nucleotides across the damage, and extend the distorted primer-template. This allows cells to preserve genetic integrity at the cost of mutations. In humans, TLS enzymes include the Y-family, inserter polymerases, Polη, Polι, Polκ, Rev1, and the B-family extender polymerase Polζ, while in S. cerevisiae only Polη, Rev1, and Polζ are present. To bypass DNA lesions, TLS polymerases cooperate, assembling into a complex on the eukaryotic sliding clamp, PCNA, termed the TLS mutasome. The mutasome assembly is contingent on protein–protein interactions (PPIs) between the modular domains and subunits of TLS enzymes, and their interactions with PCNA and DNA. While the structural mechanisms of DNA lesion bypass by the TLS polymerases and PPIs of their individual modules are well understood, the mechanisms by which they cooperate in the context of TLS complexes have remained elusive. This review focuses on structural studies of TLS polymerases and describes the case of TLS holoenzyme assemblies in action emerging from recent high-resolution Cryo-EM studies. Full article

Rev7 is a regulatory protein with roles in translesion synthesis (TLS), double strand break (DSB) repair, replication fork protection, and cell cycle regulation. Rev7 forms a homodimer in vitro using its HORMA (Hop, Rev7, Mad2) domain; however, the functional importance of Rev7 dimerization has been incompletely understood. We analyzed the functional properties of cells expressing either wild-type mouse Rev7 or Rev7^{K44A/R124A/A135D}, a mutant that cannot dimerize. The expression of wild-type Rev7, but not the mutant, rescued the sensitivity of Rev7^−/− cells to X-rays and several alkylating agents and reversed the olaparib resistance phenotype of Rev7^−/− cells. Using a novel fluorescent host-cell reactivation assay, we found that Rev7^{K44A/R124A/A135D} is unable to promote gap-filling TLS opposite an abasic site analog. The Rev7 dimerization interface is also required for shieldin function, as both Rev7^−/− cells and Rev7^−/− cells expressing Rev7^{K44A/R124A/A135D} exhibit decreased proficiency in rejoining some types of double strand breaks, as well as increased homologous recombination. Interestingly, Rev7^{K44A/R124A/A135D} retains some function in cell cycle regulation, as it maintains an interaction with Ras-related nuclear protein (Ran) and partially rescues the formation of micronuclei. The mutant Rev7 also rescues the G2/M accumulation observed in Rev7^−/− cells but does not affect progression through mitosis following nocodazole release. We conclude that while Rev7 dimerization is required for its roles in TLS, DSB repair, and regulation of the anaphase promoting complex, dimerization is at least partially dispensable for promoting mitotic spindle assembly through its interaction with Ran. Full article

DNA polymerase (pol) η is responsible for error-free translesion DNA synthesis (TLS) opposite ultraviolet light (UV)-induced cis-syn cyclobutane thymine dimers (CTDs) and cisplatin-induced intrastrand guanine crosslinks. POLH deficiency causes one form of the skin cancer-prone disease xeroderma pigmentosum variant (XPV) and cisplatin sensitivity, but the functional impacts of its germline variants remain unclear. We evaluated the functional properties of eight human POLH germline in silico-predicted deleterious missense variants, using biochemical and cell-based assays. In enzymatic assays, utilizing recombinant pol η (residues 1—432) proteins, the C34W, I147N, and R167Q variants showed 4- to 14-fold and 3- to 5-fold decreases in specificity constants (k_cat/K_m) for dATP insertion opposite the 3’-T and 5′-T of a CTD, respectively, compared to the wild-type, while the other variants displayed 2- to 4-fold increases. A CRISPR/Cas9-mediated POLH knockout increased the sensitivity of human embryonic kidney 293 cells to UV and cisplatin, which was fully reversed by ectopic expression of wild-type pol η, but not by that of an inactive (D115A/E116A) or either of two XPV-pathogenic (R93P and G263V) mutants. Ectopic expression of the C34W, I147N, and R167Q variants, unlike the other variants, did not rescue the UV- and cisplatin-sensitivity in POLH-knockout cells. Our results indicate that the C34W, I147N, and R167Q variants—substantially reduced in TLS activity—failed to rescue the UV- and cisplatin-sensitive phenotype of POLH-deficient cells, which also raises the possibility that such hypoactive germline POLH variants may increase the individual susceptibility to UV irradiation and cisplatin chemotherapy. Full article

Environmental, endogenous and therapeutic alkylating agents can react with internucleotide phosphate groups in DNA to yield alkyl phosphotriester (PTE) adducts. Alkyl-PTEs are induced at relatively high frequencies and are persistent in mammalian tissues; however, their biological consequences in mammalian cells have not been examined. Herein, we assessed how alkyl-PTEs with different alkyl group sizes and stereochemical configurations (S_P and R_P diastereomers of Me and nPr) affect the efficiency and fidelity of transcription in mammalian cells. We found that, while the R_P diastereomer of Me- and nPr-PTEs constituted moderate and strong blockages to transcription, respectively, the S_P diastereomer of the two lesions did not appreciably perturb transcription efficiency. In addition, none of the four alkyl-PTEs induced mutant transcripts. Furthermore, polymerase η assumed an important role in promoting transcription across the S_P-Me-PTE, but not any of other three lesions. Loss of other translesion synthesis (TLS) polymerases tested, including Pol κ, Pol ι, Pol ξ and REV1, did not alter the transcription bypass efficiency or mutation frequency for any of the alkyl-PTE lesions. Together, our study provided important new knowledge about the impact of alkyl-PTE lesions on transcription and expanded the substrate pool of Pol η in transcriptional bypass. Full article

DNA polymerase ζ (pol ζ) plays a central role in replicating damaged genomic DNA. When DNA synthesis stalls at a lesion, it participates in translesion DNA synthesis (TLS), which helps replication proceed. TLS prevents cell death at the expense of new mutations. The current model indicates that pol ζ-dependent TLS events are mediated by Pol31/Pol32 pol ζ subunits, which are shared with replicative polymerase pol δ. Surprisingly, we found that the mutant rev3-ΔC in yeast, which lacks the C-terminal domain (CTD) of the catalytic subunit of pol ζ and, thus, the platform for interaction with Pol31/Pol32, retains most pol ζ functions. To understand the underlying mechanisms, we studied TLS in normal templates or templates with abasic sites in vitro in primer extension reactions with purified four-subunit pol ζ versus pol ζ with Rev3-ΔC. We also examined the specificity of ultraviolet radiation (UVR)-induced mutagenesis in the rev3-ΔC strains. We found that the absence of Rev3 CTD reduces activity levels, but does not alter the basic biochemical properties of pol ζ, and alters the mutation spectrum only at high doses of UVR, alluding to the existence of mechanisms of recruitment of pol ζ to UVR-damaged sites independent of the interaction of Pol31/Pol32 with the CTD of Rev3. Full article

Translesion synthesis (TLS) is a cell signaling pathway that facilitates the tolerance of replication stress. Increased TLS activity, the particularly elevated expression of TLS polymerases, has been linked to resistance to cancer chemotherapeutics and significantly altered patient outcomes. Building upon current knowledge, we found that the expression of one of these TLS polymerases (POLI) is associated with significant differences in cervical and pancreatic cancer survival. These data led us to hypothesize that POLI expression is associated with cancer survival more broadly. However, when cancers were grouped cancer type, POLI expression did not have a significant prognostic value. We presented a binary cancer random forest classifier using 396 genes that influence the prognostic characteristics of POLI in cervical and pancreatic cancer selected via graphical least absolute shrinkage and selection operator. The classifier was then used to cluster patients with bladder, breast, colorectal, head and neck, liver, lung, ovary, melanoma, stomach, and uterus cancer when high POLI expression was associated with worsened survival (Group I) or with improved survival (Group II). This approach allowed us to identify cancers where POLI expression is a significant prognostic factor for survival (p = 0.028 in Group I and p = 0.0059 in Group II). Multiple independent validation approaches, including the gene ontology enrichment analysis and visualization tool and network visualization support the classification scheme. The functions of the selected genes involving mitochondrial translational elongation, Wnt signaling pathway, and tumor necrosis factor-mediated signaling pathway support their association with TLS and replication stress. Our multidisciplinary approach provides a novel way of identifying tumors where increased TLS polymerase expression is associated with significant differences in cancer survival. Full article

The spontaneous depurination of genomic DNA occurs frequently and generates apurinic/pyrimidinic (AP) site damage that is mutagenic or lethal to cells. Error-prone DNA polymerases are specifically responsible for the translesion synthesis (TLS) of specific DNA damage, such as AP site damage, generally with relatively low fidelity. The Y-family DNA polymerases are the main error-prone DNA polymerases, and they employ three mechanisms to perform TLS, including template-skipping, dNTP-stabilized misalignment, and misincorporation-misalignment. The bypass mechanism of the dinB homolog (Dbh), an archaeal Y-family DNA polymerase from Sulfolobus acidocaldarius, is unclear and needs to be confirmed. In this study, we show that the Dbh primarily uses template skipping accompanied by dNTP-stabilized misalignment to bypass AP site analogs, and the incorporation of the first nucleotide across the AP site is the most difficult. Furthermore, based on the reported crystal structures, we confirmed that three conserved residues (Y249, R333, and I295) in the little finger (LF) domain and residue K78 in the palm subdomain of the catalytic core domain are very important for TLS. These results deepen our understanding of how archaeal Y-family DNA polymerases deal with intracellular AP site damage and provide a biochemical basis for elucidating the intracellular function of these polymerases. Full article

DNA replication can encounter blocking obstacles, leading to replication stress and genome instability. There are several mechanisms for evading this blockade. One mechanism consists of repriming ahead of the obstacles, creating a new starting point; in humans, PrimPol is responsible for carrying out this task. PrimPol is a primase that operates in both the nucleus and mitochondria. In contrast with conventional primases, PrimPol is a DNA primase able to initiate DNA synthesis de novo using deoxynucleotides, discriminating against ribonucleotides. In vitro, PrimPol can act as a DNA primase, elongating primers that PrimPol itself sythesizes, or as translesion synthesis (TLS) DNA polymerase, elongating pre-existing primers across lesions. However, the lack of evidence for PrimPol polymerase activity in vivo suggests that PrimPol only acts as a DNA primase. Here, we provide a comprehensive review of human PrimPol covering its biochemical properties and structure, in vivo function and regulation, and the processes that take place to fill the gap-containing lesion that PrimPol leaves behind. Finally, we explore the available data on human PrimPol expression in different tissues in physiological conditions and its role in cancer. Full article

DNA-damaging chemotherapy agents such as cisplatin have been the first line of treatment for cancer for decades. While chemotherapy can be very effective, its long-term success is often reduced by intrinsic and acquired drug resistance, accompanied by chemotherapy-resistant secondary malignancies. Although the mechanisms causing drug resistance are quite distinct, they are directly connected to mutagenic translesion synthesis (TLS). The TLS pathway promotes DNA damage tolerance by supporting both replication opposite to a lesion and inaccurate single-strand gap filling. Interestingly, inhibiting TLS reduces both cisplatin resistance and secondary tumor formation. Therefore, TLS targeting is a promising strategy for improving chemotherapy. MAD2L2 (i.e., Rev7) is a central protein in TLS. It is an essential component of the TLS polymerase zeta (ζ), and it forms a regulatory complex with Rev1 polymerase. Here we present the discovery of two small molecules, c#2 and c#3, that directly bind both in vitro and in vivo to MAD2L2 and influence its activity. Both molecules sensitize lung cancer cell lines to cisplatin, disrupt the formation of the MAD2L2-Rev1 complex and increase DNA damage, hence underlining their potential as lead compounds for developing novel TLS inhibitors for improving chemotherapy treatments. Full article

Cancer therapy resistance is a persistent clinical challenge. Recently, inhibition of the mutagenic translesion synthesis (TLS) protein REV1 was shown to enhance tumor cell response to chemotherapy by triggering senescence hallmarks. These observations suggest REV1’s important role in determining cancer cell response to chemotherapy. Whether REV1 inhibition would similarly sensitize cancer cells to radiation treatment is unknown. This study reports a lack of radiosensitization in response to REV1 inhibition by small molecule inhibitors in ionizing radiation-exposed cancer cells. Instead, REV1 inhibition unexpectedly triggers autophagy, which is a known biomarker of radioresistance. We report a possible role of the REV1 TLS protein in determining cancer treatment outcomes depending upon the type of DNA damage inflicted. Furthermore, we discover that REV1 inhibition directly triggers autophagy, an uncharacterized REV1 phenotype, with a significant bearing on cancer treatment regimens. Full article

DNA lesions escaping from repair often block the DNA replicative polymerases required for DNA replication and are handled during the S/G2 phases by the DNA damage tolerance (DDT) mechanisms, which include the error-prone translesion synthesis (TLS) and the error-free template switching (TS) pathways. Where the mono-ubiquitylation of PCNA K164 is critical for TLS, the poly-ubiquitylation of the same residue is obligatory for TS. However, it is not known how cells divide the labor between TLS and TS. Due to the fact that the type of DNA lesion significantly influences the TLS and TS choice, we propose that, instead of altering the ratio between the mono- and poly-Ub forms of PCNA, the competition between TLS and TS would automatically determine the selection between the two pathways. Future studies, especially the single integrated lesion “i-Damage” system, would elucidate detailed mechanisms governing the choices of specific DDT pathways. Full article