DNA Polymerase Delta Research Articles

Targeting POLD1 to suppress the proliferation and migration of breast cancer MDA-MB-231 cell lines by downregulation of SIRT1.

The resistant and aggressive nature of triple-negative breast cancer (TNBC) renders it mostly incurable even following extensive multimodal treatment. Therefore, more studies are required to understand the underlying molecular mechanisms of its pathogenesis. SIRT1 is a class III histone deacetylase NAD+-dependent enzyme that is interlinked in tumor progression, apoptosis, metastasis, and other mechanisms of tumorigenesis, while DNA polymerase delta 1 (POLD1) functions as a gene coding for p125, which plays an important role in genome stability and DNA replication. We aimed to investigate the downstream signaling pathway of EX-527, a potent and selective SIRT1 inhibitor, in MDA-MB-231 breast cancer cell lines, and the crosstalk between SIRT1 and POLD1, which is essential for the activities of polymerase δ. The antiproliferative and apoptotic effects of EX-527 on MDA-MB-231 cells were assessed by MTT and annexin V/PI double staining assays. Migration and invasion activity of MDA-MB-231 cells were assessed by wound-healing scratch and transwell assays. Protein expressions were examined using Western Blot analysis. MDA-MB-231 cells treatment with IC50 values of 45.3μM EX-527 significantly suppressed cell proliferation and induced apoptosis by down-regulating SIRT1. Also, it significantly repressed migration and invasion of MDA-MB-231 cells as evaluated by wound healing and transwell invasion assays. Western blot results showed that decreased expression of SIRT1 is positively correlated with expression of p53 along with down-regulating POLD1. SIRT1 could have an oncogenic role in breast cancer development and progression via activating POLD1. These conclusions present new insights into the underlying mechanisms of TNBC.

DNA polymerase delta governs parental histone transfer to DNA replication lagging strand

Chromatin replication is intricately intertwined with the recycling of parental histones to the newly duplicated DNA strands for faithful genetic and epigenetic inheritance. The transfer of parental histones occurs through two distinct pathways: leading strand deposition, mediated by the DNA polymerase ε subunits Dpb3/Dpb4, and lagging strand deposition, facilitated by the MCM helicase subunit Mcm2. However, the mechanism of the facilitation of Mcm2 transferring parental histones to the lagging strand while moving along the leading strand remains unclear. Here, we show that the deletion of Pol32, a nonessential subunit of major lagging-strand DNA polymerase δ, results in a predominant transfer of parental histone H3-H4 to the leading strand during replication. Biochemical analyses further demonstrate that Pol32 can bind histone H3-H4 both in vivo and in vitro. The interaction of Pol32 with parental histone H3-H4 is disrupted through the mutation of the histone H3-H4 binding domain within Mcm2. Our findings identify the DNA polymerase δ subunit Pol32 as a critical histone chaperone downstream of Mcm2, mediating the transfer of parental histones to the lagging strand during DNA replication.

Extrachromosomal telomere DNA derived from excessive strand displacements

Alternative lengthening of telomeres (ALT) is a telomere maintenance mechanism mediated by break-induced replication, evident in approximately 15% of human cancers. A characteristic feature of ALT cancers is the presence of C-circles, circular single-stranded telomeric DNAs composed of C-rich sequences. Despite the fact that extrachromosomal C-rich single-stranded DNAs (ssDNAs), including C-circles, are unique to ALT cells, their generation process remains undefined. Here, we introduce a method to detect single-stranded telomeric DNA, called 4SET (Strand-Specific Southern-blot for Single-stranded Extrachromosomal Telomeres) assay. Utilizing 4SET, we are able to capture C-rich single-stranded DNAs that are near 200 to 1500 nucleotides in size. Both linear C-rich ssDNAs and C-circles are abundant in the fractions of cytoplasm and nucleoplasm, which supports the idea that linear and circular C-rich ssDNAs are generated concurrently. We also found that C-rich ssDNAs originate during Okazaki fragment processing during lagging strand DNA synthesis. The generation of C-rich ssDNA requires CST-PP (CTC1/STN1/TEN1-PRIMASE-Polymerase alpha) complex-mediated priming of the C-strand DNA synthesis and subsequent excessive strand displacement of the C-rich strand mediated by the DNA Polymerase delta and the BLM helicase. Our work proposes a model for the generation of C-rich ssDNAs and C-circles during ALT-mediated telomere elongation.

POLD1 Is Required for Cell Cycle Progression by Overcoming DNA Damage in Malignant Pleural Mesothelioma.

The prognosis of patients with malignant pleural mesothelioma (MPM) remains poor due to lack of effective therapeutic targets. DNA damage caused by long-time exposure to asbestos fibers has been associated with the development of MPM, with mutations at genes encoding DNA damage repair (DDR)-related molecules frequently expressed in patients with MPM. The present study was designed to identify novel therapeutic targets in MPM using large public databases, such as The Cancer Genome Atlas (TCGA) and Genotype Tissue Expression project (GTEx) focused on DDR pathways. The correlations between mRNA expression levels of DDR-related genes and overall survival (OS) were analyzed in mesothelioma patients in TCGA mesothelioma (TCGA-MESO) datasets. The anti-tumor effects of small interfering RNAs (siRNA) against DDR-related genes associated with OS were subsequently tested in MPM cell lines. High levels of mRNA encoding DNA polymerase delta 1, catalytic subunit (POLD1) were significantly associated with reduced OS in patients with MPM (p<0.001, Log-rank test). In addition, siRNA targeting POLD1 (siPOLD1) caused cell cycle arrest at the G1/S checkpoint and induced apoptosis involving accumulation of DNA damage in MPM cell lines. POLD1 plays essential roles in overcoming DNA damage and cell cycle progression at the G1/S checkpoint in MPM cells. These findings suggest that POLD1 may be a novel therapeutic target in MPM.

Human Autosomal Recessive DNA Polymerase Delta 3 Deficiency Presenting as Omenn Syndrome.

The DNA polymerase δ complex (PolD), comprising catalytic subunit POLD1 and accessory subunits POLD2, POLD3, and POLD4, is essential for DNA synthesis and is central to genome integrity. We identified, by whole exome sequencing, a homozygous missense mutation (c.1118A > C; p.K373T) in POLD3 in a patient with Omenn syndrome. The patient exhibited severely decreased numbers of naïve T cells associated with a restricted T-cell receptor repertoire and a defect in the early stages of TCR recombination. The patient received hematopoietic stem cell transplantation at age 6months. He manifested progressive neurological regression and ultimately died at age 4years. We performed molecular and functional analysis of the mutant POLD3 and assessed cell cycle progression as well as replication-associated DNA damage. Patient fibroblasts showed a marked defect in S-phase entry and an enhanced number of double-stranded DNA break-associated foci despite normal expression levels of PolD components. The cell cycle defect was rescued by transduction with WT POLD3. This study validates autosomal recessive POLD3 deficiency as a novel cause of profound T-cell deficiency and Omenn syndrome.

POLD1 DEDD Motif Mutation Confers Hypermutation in Endometrial Cancer and Durable Response to Pembrolizumab.

Mutations in the DNA polymerase delta 1 (POLD1) exonuclease domain cause DNA proofreading defects, hypermutation, hereditary colorectal and endometrial cancer, and are predictive of immunotherapy response. Exonuclease activity is carried out by two magnesium cations, bound to four highly conserved, negatively charged amino acids (AA) consisting of aspartic acid at amino acid position 316 (p.D316), glutamic acid at position 318 (p.E318), p.D402, and p.D515 (termed DEDD motif). Germline polymorphisms resulting in charge-discordant AA substitutions in the DEDD motif are classified as variants of uncertain significance (VUSs) by laboratories and thus would be considered clinically inactionable. We hypothesize this mutation class is clinically pathogenic. A review of clinical presentation was performed in our index patient with a POLD1(p.D402N) heterozygous proband with endometrial cancer. Implications of this mutation class were evaluated by a Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA)-guided systematic review, in silico analysis with orthogonal biochemical confirmation, and whole-exome and RNA sequencing analysis of the patient's tumor and engineered cell lines. Our systematic review favored a Mendelian disease mutation class associated with endometrial and colorectal cancers. In silico analysis predicted defective protein function, confirmed by biochemical assay demonstrating loss of nuclease activity. A POLD1-specific mutational signature was found in both the patient's tumor and POLD1(p.D402N) overexpressing cell. Furthermore, paired whole-exome/transcriptome analysis of endometrial tumor demonstrated hypermutation and T cell-inflamed gene expression profile (GEP), which are joint predictive biomarkers for pembrolizumab. Our patient showed a deep, durable response to immune checkpoint inhibitor (ICI). Charge-discordant AA substitution in the DEDD motif of POLD1 is detrimental to DNA proofreading and should be reclassified as likely pathogenic and possibly predictive of ICI sensitivity.

Therapeutic targets for endometriosis: Genome-wide Mendelian randomization and colocalization analyses

BackgroundEndometriosis (EM) greatly affects women's reproductive health, identifying new drug targets for EM is urgently needed. This study utilizes comprehensive genome-wide Mendelian randomization (MR) and colocalization analyses, using genomic data, to identify potential therapeutic approaches for EM. MethodsGenome-wide cis-expression quantitative trait loci (cis-eQTL) data were obtained from GTEx V8, which included 838 participants across 49 tissues or cells, and the eQTLGen consortium, which included 31,684 participants. Genome-wide association analysis (GWAS) data for EM were sourced from the FinnGen study, which consisted of 8,288 cases and 68,969 controls, as well as the UK Biobank study, which included 1,496 cases and 359,698 controls. This study utilized MR analysis to assess the correlation between genes and the risk of EM. Subsequently, colocalization analysis was conducted to investigate potential shared causal variants between the identified genes and EM. ResultsAfter conducting MR and colocalization analyses, we identified a total of 13 genes that showed significant evidence of colocalization. These genes are considered promising therapeutic candidates for treating EM. Among them, inner membrane mitochondrial protein (IMMT), src kinase associated phosphoprotein 1 (SKAP1), lysine methyltransferase 5A (KMT5A), KLF transcription factor 12 (KLF12), GRB10 interacting GYF protein 1 (GIGYF1), Wnt family member 7A (WNT7A), Sad1 and UNC84 domain containing 1 (SUN1), and poly (ADP-ribose) polymerase family member 3 (PARP3) were found to have positive associations with the risk of EM. On the other hand, progestin and adipoQ receptor family member 8 (PAQR8), adaptor related protein complex 3 subunit mu 1 (AP3M1), surfeit 6 (SURF6), TUB bipartite transcription factor (TUB), and DNA polymerase delta interacting protein 2 (POLDIP2) were found to have inverse relationships with the risk of EM. ConclusionsThrough genome-wide MR studies, a comprehensive set of genes associated with EM has been identified. Among them, IMMT, PAQR8, SKAP1, KMT5A, AP3M1, SURF6, KLF12, GIGYF1, TUB, WNT7A, SUN1, POLDIP2, and PARP3 show potential as therapeutic targets for EM treatment. Nonetheless, it is crucial to conduct further rigorous investigations to validate these prospects.

Broad antagonism of coronaviruses nsp5 to evade the host antiviral responses by cleaving POLDIP3.

Coronaviruses (CoVs) are a family of the largest RNA viruses that typically cause respiratory, enteric, and hepatic diseases in animals and humans, imposing great threats to the public safety and animal health. Porcine deltacoronavirus (PDCoV), a newly emerging enteropathogenic coronavirus, causes severe diarrhea in suckling piglets all over the world and poses potential risks of cross-species transmission. Here, we use PDCoV as a model of CoVs to illustrate the reciprocal regulation between CoVs infection and host antiviral responses. In this study, downregulation of DNA polymerase delta interacting protein 3 (POLDIP3) was confirmed in PDCoV infected IPEC-J2 cells by isobaric tags for relative and absolute quantification (iTRAQ) and Western blotting analysis. Overexpression of POLDIP3 inhibits PDCoV infection, whereas POLDIP3 knockout (POLDIP3-/-) by CRISPR-Cas9 editing significantly promotes PDCoV infection, indicating POLDIP3 as a novel antiviral regulator against PDCoV infection. Surprisingly, an antagonistic strategy was revealed that PDCoV encoded nonstructural protein 5 (nsp5) was responsible for POLDIP3 reduction via its 3C-like protease cleavage of POLDIP3 at the glutamine acid 176 (Q176), facilitating PDCoV infection due to the loss of antiviral effects of the cleaved fragments. Consistent with the obtained data in IPEC-J2 cell model in vitro, POLDIP3 reduction by cleavage was also corroborated in PDCoV infected-SPF piglets in vivo. Collectively, we unveiled a new antagonistic strategy evolved by PDCoV to counteract antiviral innate immunity by nsp5-mediated POLDIP3 cleavage, eventually ensuring productive virus replication. Importantly, we further demonstrated that nsp5s from PEDV and TGEV harbor the conserved function to cleave porcine POLDIP3 at the Q176 to despair POLDIP3-mediated antiviral effects. In addition, nsp5 from SARS-CoV-2 also cleaves human POLDIP3. Therefore, we speculate that coronaviruses employ similar POLDIP3 cleavage mechanisms mediated by nsp5 to antagonize the host antiviral responses to sustain efficient virus infection.

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.

Comparative Analysis of Cluster of Differentiation 57 and Proliferating Cell Nuclear Antigen Expression in Different Grades of Oral Squamous Cell Carcinoma: An Immunohistochemical Study.

The immune defense against tumor cells is mainly mediated by the natural killer (NK) cells. Cluster of differentiation 57 (CD57) is a 110-kd glycoprotein, typically expressed by the NK cells, attacks the cancer cells and inhibits tumor development. Proliferating cell nuclear antigen (PCNA) is a 36-kd auxiliary protein for DNA polymerase delta that correlates with cell proliferation and DNA synthesis. It is an essential component of DNA replication, DNA recombination, and DNA repair. The uncoordinated proliferation of PCNA protein characterizes the biological behavior of malignant lesions. The aim of the present study is to compare and correlate the expression of CD57 and PCNAin different grades of oral squamous cell carcinoma (OSCC) by immunohistochemistry. This retrospective analysis comprises 30 samples of various grades of OSCCsand10 samples of healthy mucosa.Sections of 4-5 µm thickness were done and stained with monoclonal anti-PCNA and anti-CD57 antibodies.The statistical package for social science (SPSS) version 16.0 software (IBM Corp., Armonk, NY) was used to analyze the data in this study. The expression of CD57 and PCNA was compared and correlated between the groups using analysis of variance (ANOVA) post hoc, Dunnet t-test, and Pearson's correlation coefficient test.For statistical significance, a p-value of 0.05 or less was used. A significant decrease in CD57 labeling index was seen from well-differentiated squamous cell carcinoma (16.63 ± 2.33) to poorly differentiated squamous cell carcinoma (5.53 ± 1.20) whereas the significant increase in PCNA labeling index was noted from well-differentiatedsquamous cell carcinoma (45.88 ± 2.20), followed by moderately differentiated and poorly differentiated squamous cell carcinoma (72.77 ± 4.35). The combination of CD57 and PCNA biomarkers appears to be good indicators of the immune status of the patient and the aggressiveness of the lesion.

DNA polymerase delta (POLD1 and POLD2) gene expression in pediatric acute lymphoblastic leukemia patients and its relationship with prognosis

Purpose: This study aimed to investigate the status of DNA polymerase delta (POLD1 and POLD2) gene expression at the time of diagnosis in pediatric acute lymphoblastic leukemia (ALL) patients, compared with the normal population, and its relationship with prognosis and other clinical findings.&#x0D; Materials and Methods: Seventy-three patients diagnosed with ALL between January 2008 and November 2015 and 29 healthy control subjects were included in the study. Gene expression profiling of peripheral blood samples was performed using Real-time PCR.&#x0D; Results: The mean value of POLD1 gene expression was found to be significantly higher in ALL patients at the time of diagnosis than the control group (376.5± 685.8 and 17.9± 19.8, respectively), but there was no difference in POLD2 gene expression (511.5± 898.1 and 125.4± 132.7, respectively). POLD1 and POLD2 gene expressions were found to be low in patients with relapse and exitus, but the results were not statistically significant. Patients with low levels of POLD1 expression had lower survival rates in the 5th year than those with high levels of expression (54% and 68%, respectively), and similarly, patients with low levels of POLD2 expression had lower survival rates in the 5th year compared to those with high levels of expression (58% and 68%, respectively).&#x0D; Conclusion: Lower POLD1 and POLD2 expressions at the time of diagnosis in ALL patients may adversely affects the prognosis.

Integrating POLE/POLD1 mutated for immunotherapy treatment planning of advanced stage non-small cell lung cancer.

In this study, we evaluated the potential of DNA polymerase epsilon (POLE) and DNA polymerase delta 1 (POLD1) as prognostic biomarkers for immune checkpoint inhibitor (ICI) treatment in patients with advanced stage non-small cell lung cancer (NSCLC). Disease stage, PD-L1 positivity, histological subtypes, POLE/POLD1 mutation status, tumor mutation burden (TMB), and response to ICIs in NSCLC cases were derived from AACR GENIE dataset (n = 24 120), TCGA-Pan Lung Cancer dataset (n = 1144), AACR GENIE BPC NSCLC v2.0-public (n = 2004), and Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets dataset (n = 350). The smoking history from TCGA and AACR GENIE datasets was grouped into current, former or never-smokers. POLE and POLD1 genetic alterations were identified in 5% and 2.6% of NSCLC patients, respectively. Current smokers had 9% and 4% of POLE/POLD1 mutations, respectively, versus 1.7% for both POLE and POLD1 mutations prevalence in never-smokers. POLE/POLD1 mutations were associated with elevated mutation counts than those with wild-type (median mutation counts 16 vs. 7, p < 0.0001), more advanced disease stages (stage I disease 15.19% vs. 29.42%), more prevalent squamous histology subtype (21.69% vs. 9.05%, p = 0.0427), and a higher percentage of PD-L1 positivity (66.67% vs. 43.87%, p < 0.001). Treatment with ICIs improved survival in patients with both POLE/POLD1 mutated and those with TMB > 18 (p < 0.001). Current smokers have a five-fold increased risk of having POLE mutations than never-smokers. POLE/POLD1 mutation status and TMB > 18 can be a composite biomarker for selecting NSCLC patients with survival benefits to ICI treatment.

DNA polymerase δ: A single Pol31 polymorphism suppresses the strain background-specific lethality of Pol32 inactivation in Saccharomyces cerevisiae

The evolutionarily conserved DNA polymerase delta (Polδ) plays several essential roles in eukaryotic DNA replication and repair, responsible for the synthesis of the lagging-strand, lower replicative mutagenesis via its proof-reading exonuclease activity and synthetizes both strands during break-induced replication. In Saccharomyces cerevisiae, the Polδ protein complex consists of three subunits encoded by the POL3, POL31 and POL32 genes. Surprisingly, in contrast to POL3 and POL31, the POL32 gene deletion was found to be viable but lethal in all other eukaryotes, raising the question to which extent the viability of the POL32 deletion in S. cerevisiae was species specific. To address this issue, we inactivated the POL32 gene in 10 evolutionary close or distant S. cerevisiae strains and found that POL32 was either essential (3 strains including SK1), non-essential (5 strains including the reference S288C strain) or confers a slow-growth phenotype (2 strains). Whole-genome sequencing of S288C/SK1 pol32∆ meiotic segregants identified the lethal/suppressor effect of the single Pol31-C43Y polymorphism. Consistently, the introduction of the Pol31–43C allele in the SK1 and West African (WA) pol32∆ mutants was sufficient to restore cell viability and wild-type growth upon introduction of two copies of POL31–43C in the SK1 haploid strain. Reciprocally, introduction of the SK1 POL31–43Y allele in the S288C pol32∆ mutant was lethal. Sequence analyses of the POL31 polymorphisms in the 1,011 yeasts genome dataset correlates with the strict occurrence of the POL31–43Y allele in the yeast African palm wine clade. Differently, the single Pol31-E400G polymorphism confers pol32∆ lethality in the Malaysian strain. In the yeast two-hybrid assay, we observed a weakened interaction between Pol3 and Pol31–43Y versus Pol31–43C suggesting an insufficient level of the Polδ holoenzyme stability/activity. Thus, the enigmatic non-essentiality of Pol32 in S. cerevisiae results from single Pol31 amino acid polymorphism and is clade rather than species specific.

The DHX9 helicase interacts with human DNA polymerase δ4 and stimulates its activity in D-loop extension synthesis

The extension of the invading strand within a displacement loop (D-loop) is a key step in homology directed repair (HDR) of doubled stranded DNA breaks. The primary goal of these studies was to test the hypotheses that 1) D-loop extension by human DNA polymerase δ4 (Pol δ4) is facilitated by DHX9, a 3′ to 5′ motor helicase, which acts to unwind the leading edge of the D-loop, and 2) the recruitment of DHX9 is mediated by direct protein-protein interactions between DHX9 and Pol δ4 and/or PCNA. DNA synthesis by Pol δ4 was analyzed in a reconstitution assay by the extension of a 93mer oligonucleotide inserted into a plasmid to form a D-loop. Product formation by Pol δ4 was monitored by incorporation of [α-32P]dNTPs into the 93mer primer followed by denaturing gel electrophoresis. The results showed that DHX9 strongly stimulated Pol δ4 mediated D-loop extension. Direct interactions of DHX9 with PCNA, the p125 and the p12 subunits of Pol δ4 were demonstrated by pull-down assays with purified proteins. These data support the hypothesis that DHX9 helicase is recruited by Pol δ4/PCNA to facilitate D-loop synthesis in HDR, and is a participant in cellular HDR. The involvement of DHX9 in HDR represents an important addition to its multiple cellular roles. Such helicase-polymerase interactions may represent an important aspect of the mechanisms involved in D-loop primer extension synthesis in HDR.

MRNA markers associated with malignant pleural effusion

Malignant pleural effusions (MPE) commonly result from malignant tumors and represent advanced-stage cancers. Thus, in clinical practice, early recognition of MPE is valuable. However, the current diagnosis of MPE is based on pleural fluid cytology or histologic analysis of pleural biopsies with a low diagnostic rate. This research aimed to assess the diagnostic ability of eight previously identified Non-Small Cell Lung Cancer (NSCLC)-associated genes for MPE. In the study, eighty-two individuals with pleural effusion were recruited. There were thirty-three patients with MPE and forty-nine patients with benign transudate. mRNA was isolated from the pleural effusion and amplified by Quantitative real-time PCR. The logistic models were further applied to evaluate the diagnostic performance of those genes. Four significant MPE-associated genes were discovered in our study, including Dual-specificity phosphatase 6 (DUSP6), MDM2 proto-oncogene (MDM2), Ring finger protein 4 (RNF4), and WEE1 G2 Checkpoint Kinase (WEE1). Pleural effusion with higher expression levels of MDM2 and WEE1 and lower expression levels of RNF4 and DUSP6 had a higher possibility of being MPE. The four-gene model had an excellent performance distinguishing MPE and benign pleural effusion, especially for pathologically negative effusions. Therefore, the gene combination is a suitable candidate for MPE screening in patients with pleural effusion. We also identified three survival-associated genes, WEE1, Neurofibromin 1 (NF1), and DNA polymerase delta interacting protein 2 (POLDIP2), which could predict the overall survival of patients with MPE.

POLD1 as a Prognostic Biomarker Correlated with Cell Proliferation and Immune Infiltration in Clear Cell Renal Cell Carcinoma.

DNA polymerase delta 1 catalytic subunit (POLD1) plays a vital role in genomic copy with high fidelity and DNA damage repair processes. However, the prognostic value of POLD1 and its relationship with tumor immunity in clear cell renal cell carcinoma (ccRCC) remains to be further explored. Transcriptional data sets and clinical information were obtained from the TCGA, ICGC, and GEO databases. Differentially expressed genes (DEGs) were derived from the comparison between the low and high POLD1 expression groups in the TCGA-KIRC cohort. KEGG and gene ontology (GO) analyses were performed for those DEGs to explore the potential influence of POLD1 on the biological behaviors of ccRCC. The prognostic clinical value and mutational characteristics of patients were described and analyzed according to the POLD1 expression levels. TIMER and TISIDB databases were utilized to comprehensively investigate the potential relevance between the POLD1 levels and the status of the immune cells, as well as the tumor infiltration of immune cells. In addition, RT-qPCR, Western blot, immunohistochemistry and several functional and animal experiments were performed for clinical, in vitro and in vivo validation. POLD1 was highly expressed in a variety of tumors including ccRCC, and further verified in a validation cohort of 60 ccRCC samples and in vitro cell line experiments. POLD1 expression levels in the ccRCC samples were associated with various clinical characteristics including pathologic tumor stage and histologic grade. ccRCC patients with high POLD1 expression have poor clinical outcomes and exhibit a higher rate of somatic mutations than those with low POLD1 expression. Cox regression analysis also showed that POLD1 could act as a potential independent prognostic biomarker. The DEGs associated with POLD1 were significantly enriched in the immunity-related pathways. Moreover, further immune infiltration analysis indicated that high POLD1 expression was associated with high NK CD56bright cells, Treg cells, and myeloid-derived suppressor cells' (MDSCs) infiltration scores, as well as their marker gene sets of immune cell status. Meanwhile, POLD1 exhibited resistance to various drugs when highly expressed. Finally, the knockdown of POLD1 inhibited the proliferation and migration, and promoted the apoptosis of ccRCC cells in vitro and in vivo, as well as influenced the activation of oncogenic signaling. Our current study demonstrated that POLD1 is a potential prognostic biomarker for ccRCC patients. It might create a tumor immunosuppressive microenvironment and inhibit the susceptibility to ferroptosis leading to a poor prognosis.

DNA Polymerase Delta Exhibits Altered Catalytic Properties on Lysine Acetylation.

DNA polymerase delta is the primary polymerase that is involved in undamaged nuclear lagging strand DNA replication. Our mass-spectroscopic analysis has revealed that the human DNA polymerase δ is acetylated on subunits p125, p68, and p12. Using substrates that simulate Okazaki fragment intermediates, we studied alterations in the catalytic properties of acetylated polymerase and compared it to the unmodified form. The current data show that the acetylated form of human pol δ displays a higher polymerization activity compared to the unmodified form of the enzyme. Additionally, acetylation enhances the ability of the polymerase to resolve complex structures such as G-quadruplexes and other secondary structures that might be present on the template strand. More importantly, the ability of pol δ to displace a downstream DNA fragment is enhanced upon acetylation. Our current results suggest that acetylation has a profound effect on the activity of pol δ and supports the hypothesis that acetylation may promote higher-fidelity DNA replication.

Prospects of POLD1 in Human Cancers: A Review.

Cancer is the second leading cause of death globally, exceeded only by cardiovascular disease. Despite the introduction of several survival-prolonging treatment modalities, including targeted therapy and immunotherapy, the overall prognosis for the metastatic disease remains challenging. Therefore, the identification of new molecular biomarkers and therapeutic targets related to cancer diagnosis and prognosis is of paramount importance. DNA polymerase delta 1 (POLD1), a catalytic and proofreading subunit of the DNA polymerase δ complex, performs a crucial role in DNA replication and repair processes. Recently, germline and somatic mutations of the POLD1 gene have been acknowledged in several malignancies. Moreover, diversified POLD1 expression profiles have been reported in association with clinicopathological features in a variety of tumor types. With this review, we aim to summarize the current knowledge on the role of POLD1 in cancers. In addition, we discuss the future prospects and clinical applications of the assessment of POLD1 mutation and expression patterns in tumors.

CAF-1 deposits newly synthesized histones during DNA replication using distinct mechanisms on the leading and lagging strands.

During every cell cycle, both the genome and the associated chromatin must be accurately replicated. Chromatin Assembly Factor-1 (CAF-1) is a key regulator of chromatin replication, but how CAF-1 functions in relation to the DNA replication machinery is unknown. Here, we reveal that this crosstalk differs between the leading and lagging strand at replication forks. Using biochemical reconstitutions, we show that DNA and histones promote CAF-1 recruitment to its binding partner PCNA and reveal that two CAF-1 complexes are required for efficient nucleosome assembly under these conditions. Remarkably, in the context of the replisome, CAF-1 competes with the leading strand DNA polymerase epsilon (Polϵ) for PCNA binding. However, CAF-1 does not affect the activity of the lagging strand DNA polymerase Delta (Polδ). Yet, in cells, CAF-1 deposits newly synthesized histones equally on both daughter strands. Thus, on the leading strand, chromatin assembly by CAF-1 cannot occur simultaneously to DNA synthesis, while on the lagging strand these processes may be coupled. We propose that these differences may facilitate distinct parental histone recycling mechanisms and accommodate the inherent asymmetry of DNA replication.

Bavachinin protects the liver in NAFLD by promoting regeneration via targeting PCNA

IntroductionNonalcoholic fatty liver disease (NAFLD) is the most common liver disease all over the world, and no drug is approved for the treatment of NAFLD. Bavachinin (BVC) is proven to possess liver-protecting effect against NAFLD, but its mechanism is still blurry. ObjectivesWith the use of Click Chemistry-Activity‐Based Protein Profiling (CC-ABPP) technology, this study aims to identify the target of BVC, and investigate the mechanism by which BVC exerts its liver-protecting effect. MethodsThe high fat diet induced hamster NAFLD model is introduced to investigate BVC’s lipid-lowering and liver-protecting effects. Then, a small molecular probe ofBVC is designed and synthesized based on theCC-ABPP technology, and BVC’s target is fished out. A series of experiments are performed to identify the target, including competitive inhibition assay, surface-plasmon resonance (SPR), cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS) assay, and co-immunoprecipitation (Co-IP). Afterward, the pro-regeneration effects of BVC are validated in vitro and in vivo through flow cytometry, immunofluorescence, and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). ResultIn the hamster NAFLD model, BVC shows lipid-lowing effect and improvement on the histology. PCNA is identified as the target of BVC with the method mentioned above, and BVC facilitates the interaction between PCNA and DNA polymerase delta. BVC promotes HepG2 cells proliferation which is inhibited by T2AA, an inhibitor suppresses the interaction between PCNA and DNA polymerase delta. In NAFLD hamsters, BVC enhances PCNA expression and liver regeneration, reduces hepatocyte apoptosis. ConclusionThis study suggests that, besides the anti-lipemic effect, BVC binds to the pocket of PCNA facilitating its interaction with DNA polymerase delta and pro-regeneration effect, thereby exerts the protective effect against HFD induced liver injury.