Break-induced replication promotes fragile telomere formation.

TRF1 facilitates the replication of telomeric DNA in part by recruiting the BLM helicase, which can resolve G-quadruplexes on the lagging-strand template. Lagging-strand telomeres lacking TRF1 or BLM form fragile telomeres-structures that resemble common fragile sites (CFSs)-but how they are formed is not known. We report that analogous to CFSs, fragile telomeres in BLM-deficient cells involved double-strand break (DSB) formation, in this case by the SLX4/SLX1 nuclease. The DSBs were repaired by POLD3/POLD4-dependent break-induced replication (BIR), resulting in fragile telomeres containing conservatively replicated DNA. BIR also promoted fragile telomere formation in cells with FokI-induced telomeric DSBs and in alternative lengthening of telomeres (ALT) cells, which have spontaneous telomeric damage. BIR of telomeric DSBs competed with PARP1-, LIG3-, and XPF-dependent alternative nonhomologous end joining (alt-NHEJ), which did not generate fragile telomeres. Collectively, these findings indicate that fragile telomeres can arise from BIR-mediated repair of telomeric DSBs.

UCHL3 induces radiation resistance and acquisition of mesenchymal phenotypes by deubiquitinating POLD4 in glioma stem cells.

BACKGROUND: The high degree of intratumoral genomic heterogeneity is a major obstacle for glioblastoma (GBM) tumors, one of the most lethal human malignancies, and is thought to influence conventional therapeutic outcomes negatively. The proneural-to-mesenchymal transition (PMT) of glioma stem cells (GSCs) confers resistance to radiation therapy in glioblastoma patients. POLD4 is associated with cancer progression, while the mechanisms underlying PMT and tumor radiation resistance have remained elusive. METHOD: Expression and prognosis of the POLD family were analyzed in TCGA, the Chinese Glioma Genome Atlas (CGGA) and GEO datasets. Tumorsphere formation and in vitro limiting dilution assay were performed to investigate the effect of UCHL3-POLD4 on GSC self-renewal. Apoptosis, TUNEL, cell cycle phase distribution, modification of the Single Cell Gel Electrophoresis (Comet), γ-H2AX immunofluorescence, and colony formation assays were conducted to evaluate the influence of UCHL3-POLD4 on GSC in ionizing radiation. Coimmunoprecipitation and GST pull-down assays were performed to identify POLD4 protein interactors. In vivo, intracranial xenograft mouse models were used to investigate the molecular effect of UCHL3, POLD4 or TCID on GCS. RESULT: We determined that POLD4 was considerably upregulated in MES-GSCs and was associated with a meagre prognosis. Ubiquitin carboxyl terminal hydrolase L3 (UCHL3), a DUB enzyme in the UCH protease family, is a bona fide deubiquitinase of POLD4 in GSCs. UCHL3 interacted with, depolyubiquitinated, and stabilized POLD4. Both in vitro and in vivo assays indicated that targeted depletion of the UCHL3-POLD4 axis reduced GSC self-renewal and tumorigenic capacity and resistance to IR treatment by impairing homologous recombination (HR) and nonhomologous end joining (NHEJ). Additionally, we proved that the UCHL3 inhibitor TCID induced POLD4 degradation and can significantly enhance the therapeutic effect of IR in a gsc-derived in situ xenograft model. CONCLUSION: These findings reveal a new signaling axis for GSC PMT regulation and highlight UCHL3-POLD4 as a potential therapeutic target in GBM. TCID, targeted for reducing the deubiquitinase activity of UCHL3, exhibited significant synergy against MES GSCs in combination with radiation.

Expression Profile, Molecular Association, and Clinical Significance of POLD4 in Glioblastoma.

The POLD4 gene encodes a subunit (δ4) of DNA polymerase delta, which is a key enzyme involved in DNA replication and repair. Recent studies have suggested that POLD4 plays a crucial role in developing certain cancers. However, there is a lack of knowledge regarding the role of POLD4 in the context of glioblastoma (GBM). Therefore, in this study we have used various cancer bioinformatics tools to explore the role of POLD4 in glioblastoma. Data from various sources were accessed to analyze POLD4 gene expression and estimate tumor-infiltrating immune cells in glioblastoma. Methylation data were retrieved using the MEXPRESS web browser and analyzed. UALCAN webserver was used to analyze the protein expression of POLD4. Gene correlation and pathway enrichment analysis were performed using cBioPortal and GSEA software, respectively. Afterward, survival analysis was performed. POLD4 was significantly upregulated in glioblastoma at both gene and protein levels in GBM, and ROC curve analysis revealed it as a potential biomarker in glioblastoma. GSEA analysis of TCGA-GBM pan-cancer study exhibited that POLD4 expression was associated with critical pathways, such as interferon-gamma response, G2M checkpoint, inflammatory response, E2F targets, EMT transition, and KRAS signaling pathways. Furthermore, POLD4 expression was positively correlated with DNA methylation at 3 CpG sites, including Cg16509978, with a Pearson correlation coefficient value of 0.398 (p-value ≤ 0.01), while the promoter region had a positive correlation but was not significant. In addition, POLD4 is significantly linked with poor OS, PFS, and DFS. We also found association of POLD4 expression with altered immune cell infiltration. In conclusion, POLD4 is significantly upregulated in glioblastoma and may be used as a potential diagnostic or prognostic biomarker for GBM patients. However, to establish the same a large cohort study is needed. Using TCGA data and various cancer bioinformatics tools mentioned above we observed very high level of gene and protein expression of POLD4 in glioblastoma patients. The expression of POLD4 was significantly correlated with inflammatory and oncogenic pathways and it also has a significant correlation with adverse outcome in patients with glioblastoma.

POLD4 Promotes Glioma Cell Proliferation and Suppressive Immune Microenvironment: A Pan-Cancer Analysis Integrated with Experimental Validation.

POLD4 plays a crucial part in the complex machinery of DNA replication and repair as a vital component of the DNA polymerase delta complex. In this research, we obtained original information from various publicly available databases. Using a blend of R programming and internet resources, we initiated an extensive examination into the correlation between POLD4 expression and the various elements of cancers. In addition, we performed knockdown experiments in glioma cell lines to authenticate its significant impact. We discovered that POLD4 is upregulated in various malignant tumors, demonstrating a significant correlation with poor patient survival prognosis. Using function analysis, it was uncovered that POLD4 exhibited intricate associations with signaling pathways spanning multiple tumor types. Subsequent investigations unveiled the close association of POLD4 with the immune microenvironment and the effectiveness of immunotherapy. Drugs like trametinib, saracatinib, and dasatinib may be used in patients with high POLD4. Using experimental analysis, we further confirmed the overexpression of POLD4 in gliomas, as well as its correlation with glioma recurrence, proliferation, and the suppressive immune microenvironment. Our research findings indicate that the expression pattern of POLD4 not only serves as a robust indicator of prognosis in cancer patients but also holds promising potential as a new focus for treatment.

Quaternary structural diversity in eukaryotic DNA polymerases: monomeric to multimeric form.

Sixteen eukaryotic DNA polymerases have been identified and studied so far. Based on the sequence similarity of the catalytic subunits of DNA polymerases, these have been classified into four A, B, X and Y families except PrimPol, which belongs to the AEP family. The quaternary structure of these polymerases also varies depending upon whether they are composed of one or more subunits. Therefore, in this review, we used a quaternary structure-based classification approach to group DNA polymerases as either monomeric or multimeric and highlighted functional significance of their accessory subunits. Additionally, we have briefly summarized various DNA polymerase discoveries from a historical perspective, emphasized unique catalytic mechanism of each DNA polymerase and highlighted recent advances in understanding their cellular functions.

Alternative lengthening of human telomeres is a conservative DNA replication process with features of break-induced replication.

Human malignancies overcome replicative senescence either by activating the reverse-transcriptase telomerase or by utilizing a homologous recombination-based mechanism, referred to as alternative lengthening of telomeres (ALT). In budding yeast, ALT exhibits features of break-induced replication (BIR), a repair pathway for one-ended DNA double-strand breaks (DSBs) that requires the non-essential subunit Pol32 of DNA polymerase delta and leads to conservative DNA replication. Here, we examined whether ALT in human cancers also exhibits features of BIR A telomeric fluorescence in situ hybridization protocol involving three consecutive staining steps revealed the presence of conservatively replicated telomeric DNA in telomerase-negative cancer cells. Furthermore, depletion of PolD3 or PolD4, two subunits of human DNA polymerase delta that are essential for BIR, reduced the frequency of conservatively replicated telomeric DNA ends and led to shorter telomeres and chromosome end-to-end fusions. Taken together, these results suggest that BIR is associated with conservative DNA replication in human cells and mediates ALT in cancer.

Downregulation of POLD4 in Calu6 cells results in G1-S blockage through suppression of the Akt-Skp2-p27 pathway.

Previously, we have shown that downregulation of POLD4 in lung cancer cells delays progression through the G1-S cell cycle transition and leads to increased genomic instability. To date however, detailed molecular mechanisms have not been elucidated to explain how this occurs. In the present study, we found that reduction in POLD4 by siRNA knockdown promoted downregulation of both p-Akt Ser473 and Skp2 as well as upregulation of p27. Furthermore, these protein expression levels were rescued when siRNA-resistant POLD4 was ectopically expressed in the knockdown cells. These data suggest that the POLD4 downregulation is associated with impaired Akt-Skp2-p27 pathway in lung cancer.

Pold4 is dispensable for mouse development, DNA replication and DNA repair.

The DNA polymerase delta (Pol δ), a heterotetramer of four subunits (Pol δ4), plays a pivotal role in DNA replication, as well as in DNA damage repair. Pold4, as the smallest subunit of Pol δ, is degraded in response to DNA damage or when entering into S-phase. This leads to the conversion of Pol δ4 to the trimeric complex Pol δ3. However, the contribution of Pold4 has not been fully elucidated in mammals. Cdm1, the Pold4 ortholog in Schizosaccharomyces pombe, is dispensable for cell growth and DNA damage repair, and there are no Pold4 orthologs in Saccharomyces cerevisiae. We previously generated a knockout mouse model of Pold3 and revealed its essential role in genome stability. Unexpectedly, we here found that Pold4 knockout mice are viable and fertile. In addition, Pold4 knockout mice do not exhibit any pathologic changes in the lung and spleen, tissues with the most abundant expression of Pold4. Moreover, Pold4 knockout mouse tail tip fibroblasts (TTF) exhibited normal cell growth, cell cycle, DNA replication, DNA damage and DNA repair capacity. These results suggested that Pol δ3 but not Pol δ4 may be responsible for these processes in normal cells. Interestingly, 19-month-old wild-type (WT) mice had tumors in the liver, while Pold4 knockout mice did not, and Pold4 knockout mice showed increased longevity. In further, this provided evidence suggested that Pold4 could be a potential novel target for lung carcinoma because its depletion does not affect normal cells but does affect cancer cells.

Non-canonical binding of the Chaetomium thermophilum PolD4 N-terminal PIP motif to PCNA involves Q-pocket and compact 2-fork plug interactions but no 310 helix.

DNA polymerase δ (Pol δ) is a key enzyme for the maintenance of genome integrity in eukaryotic cells, acting in concert with the sliding clamp processivity factor PCNA (proliferating cell nuclear antigen). Three of the four subunits of human Pol δ interact directly with the PCNA homotrimer via a short, conserved protein sequence known as a PCNA interacting protein (PIP) motif. Here, we describe the identification of a PIP motif located towards the N terminus of the PolD4 subunit of Pol δ (equivalent to human p12) from the thermophilic filamentous fungus Chaetomium thermophilum and present the X-ray crystal structure of the corresponding peptide bound to PCNA at 2.45 Å. Like human p12, the fungal PolD4 PIP motif displays non-canonical binding to PCNA. However, the structures of the human p12 and fungal PolD4 PIP motif peptides are quite distinct, with the fungal PolD4 PIP motif lacking the 310 helical segment that characterises most previously identified PIP motifs. Instead, the fungal PolD4 PIP motif binds PCNA via conserved glutamine that inserts into the Q-pocket on the surface of PCNA and with conserved leucine and phenylalanine sidechains forming a compact 2-fork plug that inserts into the hydrophobic pocket on PCNA. Despite the unusual binding mode of the fungal PolD4, isothermal calorimetry (ITC) measurements show that its affinity for PCNA is similar to that of its human orthologue. These observations add to a growing body of information on how diverse proteins interact with PCNA and highlight how binding modes can vary significantly between orthologous PCNA partner proteins.

Two forms of human DNA polymerase δ: Who does what and why?

DNA polymerase δ (Pol δ) plays a central role in lagging strand DNA synthesis in eukaryotic cells, as well as an important role in DNA repair processes. Human Pol δ4 is a heterotetramer of four subunits, the smallest of which is p12. Pol δ3 is a trimeric form that is generated in vivo by the degradation of the p12 subunit in response to DNA damage, and during entry into S-phase. The biochemical properties of the two forms of Pol δ, as well as the changes in their distribution during the cell cycle, are reviewed from the perspective of understanding their respective cellular functions. Biochemical and cellular studies support a role for Pol δ3 in gap filling during DNA repair, and in Okazaki fragment synthesis during DNA replication. Recent studies of cells in which p12 expression is ablated, and are therefore null for Pol δ4, show that Pol δ4 is not required for cell viability. These cells have a defect in homologous recombination, revealing a specific role for Pol δ4 that cannot be performed by Pol δ3. Pol δ4 activity is required for D-loop displacement synthesis in HR. The reasons why Pol δ4 but not Pol δ3 can perform this function are discussed, as well as the question of whether helicase action is needed for efficient D-loop displacement synthesis. Pol δ4 is largely present in the G1 and G2/M phases of the cell cycle and is low in S phase. This is discussed in relation to the availability of Pol δ4 as an additional layer of regulation for HR activity during cell cycle progression.

4-Nitroquinoline-1-oxide effects human lung adenocarcinoma A549 cells by regulating the expression of POLD4.

The aim of the present study was to explore the expression of POLD4 in human lung adenocarcinoma A549 cells under 4-nitroquinoline-1-oxide (4NQO) stimulation to investigate the role of POLD4 in smoking-induced lung cancer. The lung cancer A549 cell line was treated with 4NQO, with or without MG132 (an inhibitor of proteasome activity), and subsequently the POLD4 level was determined by western blot analysis. Secondly, the cell sensitivity to 4NQO and Taxol was determined when the POLD4 expression level was downregulated by siRNA. The POLD4 protein levels in the A549 cells decreased following treatment with 4NQO; however, MG132 could reverse this phenotype. Downregulation of the POLD4 expression by siRNA enhanced A549 cell sensitivity to 4NQO, but not to Taxol. In conclusion, 4NQO affects human lung adenocarcinoma A549 cells by regulating the expression of POLD4.