The PCNA-Pol δ complex couples lagging strand DNA synthesis to parental histone transfer for epigenetic inheritance.

Inheritance of epigenetic information is critical for maintaining cell identity. The transfer of parental histone H3-H4 tetramers, the primary carrier of epigenetic modifications on histone proteins, represents a crucial yet poorly understood step in the inheritance of epigenetic information. Here, we show the lagging strand DNA polymerase, Pol δ, interacts directly with H3-H4 and that the interaction between Pol δ and the sliding clamp PCNA regulates parental histone transfer to lagging strands, most likely independent of their roles in DNA synthesis. When combined, mutations at Pol δ and Mcm2 that compromise parental histone transfer result in a greater reduction in nucleosome occupancy at nascent chromatin than mutations in either alone. Last, PCNA contributes to nucleosome positioning on nascent chromatin. On the basis of these results, we suggest that the PCNA-Pol δ complex couples lagging strand DNA synthesis to parental H3-H4 transfer, facilitating epigenetic inheritance.

Immune checkpoint inhibitors for POLE or POLD1 proofreading-deficient metastatic colorectal cancer.

BACKGROUND: POLE and POLD1 proofreading deficiency (POLE/D1pd) define a rare subtype of ultramutated metastatic colorectal cancer (mCRC; over 100 mut/Mb). Disease-specific data about the activity and efficacy of immune checkpoint inhibitors (ICIs) in POLE/D1pd mCRC are lacking and it is unknown whether outcomes may be different from mismatch repair-deficient (dMMR)/microsatellite instability-high (MSI-H) mCRCs treated with ICIs. PATIENTS AND METHODS: In this global study, we collected 27 patients with mCRC harboring POLE/D1 mutations leading to proofreading deficiency and treated with anti-programmed cell death-ligand 1 alone +/- anti-cytotoxic T-lymphocyte antigen-4 agents. We collected clinicopathological and genomic characteristics, response, and survival outcomes after ICIs of POLE/D1pd mCRC and compared them with a cohort of 610 dMMR/MSI-H mCRC patients treated with ICIs. Further genomic analyses were carried out in an independent cohort of 7241 CRCs to define POLE and POLD1pd molecular profiles and mutational signatures. RESULTS: POLE/D1pd was associated with younger age, male sex, fewer RAS/BRAF driver mutations, and predominance of right-sided colon cancers. Patients with POLE/D1pd mCRC showed a significantly higher overall response rate (ORR) compared to dMMR/MSI-H mCRC (89% versus 54%; P = 0.01). After a median follow-up of 24.9 months (interquartile range: 11.3-43.0 months), patients with POLE/D1pd showed a significantly superior progression-free survival (PFS) compared to dMMR/MSI-H mCRC [hazard ratio (HR) = 0.24, 95% confidence interval (CI) 0.08-0.74, P = 0.01] and superior overall survival (OS) (HR = 0.38, 95% CI 0.12-1.18, P = 0.09). In multivariable analyses including the type of DNA repair defect, POLE/D1pd was associated with significantly improved PFS (HR = 0.17, 95% CI 0.04-0.69, P = 0.013) and OS (HR = 0.24, 95% CI 0.06-0.98, P = 0.047). Molecular profiling showed that POLE/D1pd tumors have higher tumor mutational burden (TMB). Responses were observed in both subtypes and were associated with the intensity of POLE/D1pd signature. CONCLUSIONS: Patients with POLE/D1pd mCRC showed more favorable outcomes compared to dMMR/MSI-H mCRC to treatment with ICIs in terms of tumor response and survival.

Development of a new caged intein for multi-input conditional translation of synthetic mRNA.

mRNA medicines can be used to express therapeutic proteins, but the production of such proteins in non-target cells has a risk of adverse effects. To accurately distinguish between therapeutic target and nontarget cells, it is desirable to utilize multiple proteins expressed in each cell as indicators. To achieve such multi-input translational regulation of mRNA medicines, in this study, we engineered Rhodothermus marinus (Rma) DnaB intein to develop "caged Rma DnaB intein" that enables conditional reconstitution of full-length translational regulator protein from split fragments. By combining the caged Rma DnaB intein, the split translational regulator protein, and target protein-binding domains, we succeeded in target protein-dependent translational repression of mRNA in human cells. In addition, the caged Rma intein showed orthogonality to the previously reported Nostoc punctiforme (Npu) DnaE-based caged intein. Finally, by combining these two orthogonal caged inteins, we developed an mRNA-based logic gate that regulates translation based on the expression of multiple intracellular proteins. This study provides important information to develop safer mRNA medicines.

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.

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.

Pold4 subunit of replicative polymerase δ promotes fork slowing at broken templates.

Single-strand breaks (SSBs) are the most frequent type of lesion, and replication across such lesions leads to double-strand breaks (DSBs). DSBs that arise during replication are repaired by homologous recombination (HR) and are suppressed by fork reversal. Poly[ADP-ribose] polymerase I (PARP1) and the proofreading exonuclease activity of replicative polymerase ε (Polε) are required for fork reversal when leading strand replication encounters SSBs. However, the mechanism underlying fork reversal at the SSB during lagging-strand replication remains elusive. We here demonstrate that the Pold4 subunit of replicative polymerase δ (Polδ) plays a role in promoting fork reversal during lagging strand replication on a broken template. POLD4-/- cells exhibited heightened sensitivity to camptothecin (CPT) but not to other DNA-damaging agents compared to wild-type cells. This selective CPT sensitivity in POLD4-/- cells suggests that Pold4 suppresses DSBs during replication, as CPT induces significant SSBs during replication, which subsequently lead to DSBs. To explore the functional interactions among Pold4, Polε exonuclease, and PARP1 in DSB suppression, we generated PARP1-/-, POLD4-/-, Polε exonuclease-deficient POLE1exo-/-, PARP1-/-/POLD4-/-, and POLD4-/-/POLE1exo-/- cells. These epistasis analyses showed that Pold4 is involved in the PARP1-Polε exonuclease-mediated fork reversal following CPT treatment. These results suggest that Pold4 aids in fork reversal when lagging strand replication stalls on a broken template. In conclusion, the Pold4 subunit of Polδ has roles in the PARP1-Polε exonuclease-mediated fork reversal, contributing to the suppression of DSBs.

Discovery of recessive effect of human polymerase δ proofreading deficiency through mutational analysis of POLD1-mutated normal and cancer cells.

Constitutional heterozygous pathogenic variants in the exonuclease domain of POLE and POLD1, which affect the proofreading activity of the corresponding polymerases, cause a cancer predisposition syndrome characterized by increased risk of gastrointestinal polyposis, colorectal cancer, endometrial cancer and other tumor types. The generally accepted explanation for the connection between the disruption of the proofreading activity of polymerases epsilon and delta and cancer development is through an increase in the somatic mutation rate. Here we studied an extended family with multiple members heterozygous for the pathogenic POLD1 variant c.1421T>C p.(Leu474Pro), which segregates with the polyposis and cancer phenotypes. Through the analysis of mutational patterns of patient-derived fibroblasts colonies and de novo mutations obtained by parent-offspring comparisons, we concluded that heterozygous POLD1 L474P just subtly increases the somatic and germline mutation burden. In contrast, tumors developed in individuals with a heterozygous mutation in the exonuclease domain of POLD1, including L474P, have an extremely high mutation rate (>100 mut/Mb) associated with signature SBS10d. We solved this contradiction through the observation that tumorigenesis involves somatic inactivation of the wildtype POLD1 allele. These results imply that exonuclease deficiency of polymerase delta has a recessive effect on mutation rate.

Replication of [AT/TA]25 Microsatellite Sequences by Human DNA Polymerase δ Holoenzymes Is Dependent on dNTP and RPA Levels.

Fragile sites are unstable genomic regions that are prone to breakage during stressed DNA replication. Several common fragile sites (CFS) contain A+T-rich regions including perfect [AT/TA] microsatellite repeats that may collapse into hairpins when in single-stranded DNA (ssDNA) form and coincide with chromosomal hotspots for breakage and rearrangements. While many factors contribute to CFS instability, evidence exists for replication stalling within [AT/TA] microsatellite repeats. Currently, it is unknown how stress causes replication stalling within [AT/TA] microsatellite repeats. To investigate this, we utilized FRET to characterize the structures of [AT/TA]25 sequences and also reconstituted lagging strand replication to characterize the progression of pol δ holoenzymes through A+T-rich sequences. The results indicate that [AT/TA]25 sequences adopt hairpins that are unwound by the major ssDNA-binding complex, RPA, and the progression of pol δ holoenzymes through A+T-rich sequences saturated with RPA is dependent on the template sequence and dNTP concentration. Importantly, the effects of RPA on the replication of [AT/TA]25 sequences are dependent on dNTP concentration, whereas the effects of RPA on the replication of A+T-rich, nonstructure-forming sequences are independent of dNTP concentration. Collectively, these results reveal complexities in lagging strand replication and provide novel insights into how [AT/TA] microsatellite repeats contribute to genome instability.

Starting DNA Synthesis: Initiation Processes during the Replication of Chromosomal DNA in Humans.

The initiation reactions of DNA synthesis are central processes during human chromosomal DNA replication. They are separated into two main processes: the initiation events at replication origins, the start of the leading strand synthesis for each replicon, and the numerous initiation events taking place during lagging strand DNA synthesis. In addition, a third mechanism is the re-initiation of DNA synthesis after replication fork stalling, which takes place when DNA lesions hinder the progression of DNA synthesis. The initiation of leading strand synthesis at replication origins is regulated at multiple levels, from the origin recognition to the assembly and activation of replicative helicase, the Cdc45-MCM2-7-GINS (CMG) complex. In addition, the multiple interactions of the CMG complex with the eukaryotic replicative DNA polymerases, DNA polymerase α-primase, DNA polymerase δ and ε, at replication forks play pivotal roles in the mechanism of the initiation reactions of leading and lagging strand DNA synthesis. These interactions are also important for the initiation of signalling at unperturbed and stalled replication forks, "replication stress" events, via ATR (ATM-Rad 3-related protein kinase). These processes are essential for the accurate transfer of the cells' genetic information to their daughters. Thus, failures and dysfunctions in these processes give rise to genome instability causing genetic diseases, including cancer. In their influential review "Hallmarks of Cancer: New Dimensions", Hanahan and Weinberg (2022) therefore call genome instability a fundamental function in the development process of cancer cells. In recent years, the understanding of the initiation processes and mechanisms of human DNA replication has made substantial progress at all levels, which will be discussed in the review.

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

BACKGROUND/AIM: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Race, Prevalence of POLE and POLD1 Alterations, and Survival Among Patients With Endometrial Cancer.

Importance: Black patients with endometrial cancer (EC) in the United States have higher mortality than patients of other races with EC. The prevalence of POLE and POLD1 pathogenic alterations in patients of different races with EC are not well studied. Objective: To explore the prevalence of and outcomes associated with POLE and POLD1 alterations in differential racial groups. Design, Setting, and Participants: This retrospective cohort study incorporated the largest available data set of patients with EC, including American Association for Cancer Research Project GENIE (Genomics Evidence Neoplasia Information Exchange; 5087 participants), Memorial Sloan Kettering-Metastatic Events and Tropisms (1315 participants), and the Cancer Genome Atlas Uterine Corpus Endometrial Carcinoma (517 participants), collected from 2015 to 2023, 2013 to 2021, and 2006 to 2012, respectively. The prevalence of and outcomes associated with POLE or POLD1 alterations in EC were evaluated across self-reported racial groups. Exposure: Patients of different racial groups with EC and with or without POLE or POLD1 alterations. Main Outcomes and Measures: The main outcome was overall survival. Data on demographic characteristics, POLE and POLD1 alteration status, histologic subtype, tumor mutation burden, fraction of genome altered, and microsatellite instability score were collected. Results: A total of 6919 EC cases were studied, of whom 444 (6.4%), 694 (10.0%), and 4869 (70.4%) patients were self-described as Asian, Black, and White, respectively. Within these large data sets, Black patients with EC exhibited a lower weighted average prevalence of pathogenic POLE alterations (0.5% [3 of 590 cases]) compared with Asian (6.1% [26 of 424]) or White (4.6% [204 of 4520]) patients. By contrast, the prevalence of POLD1 pathogenic alterations was 5.0% (21 cases), 3.2% (19 cases), and 5.6% (255 cases) in Asian, Black, and White patients with EC, respectively. Patients with POLD1 alterations had better outcomes regardless of race, histology, and TP53 alteration status. For a total of 241 clinically annotated Black patients with EC, a composite biomarker panel of either POLD1 or POLE alterations identified 7.1% (17 patients) with positive outcomes (1 event at 70 months follow up) in the small sample of available patients. Conclusions and Relevance: In this retrospective clinicopathological study of patients of different racial groups with EC, a composite biomarker panel of either POLD1 or POLE alteration could potentially guide treatment de-escalation, which is especially relevant for Black patients.

DNA polymerase ε and δ variants drive mutagenesis in polypurine tracts in human tumors.

Alterations in the exonuclease domain of DNA polymerase ε cause ultramutated cancers. These cancers accumulate AGA>ATA transversions; however, their genomic features beyond the trinucleotide motifs are obscure. We analyze the extended DNA context of ultramutation using whole-exome sequencing data from 524 endometrial and 395 colorectal tumors. We find that G>T transversions in POLE-mutant tumors predominantly affect sequences containing at least six consecutive purines, with a striking preference for certain positions within polypurine tracts. Using this signature, we develop a machine-learning classifier to identify tumors with hitherto unknown POLE drivers and validate two drivers, POLE-E978G and POLE-S461L, by functional assays in yeast. Unlike other pathogenic variants, the E978G substitution affects the polymerase domain of Pol ε. We further show that tumors with POLD1 drivers share the extended signature of POLE ultramutation. These findings expand the understanding of ultramutation mechanisms and highlight peculiar mutagenic properties of polypurine tracts in the human genome.

Double-strand breaks induce inverted duplication chromosome rearrangements by a DNA polymerase δ-dependent mechanism.

Inverted duplications, also known as foldback inversions, are commonly observed in cancers and are the major class of chromosome rearrangement recovered from yeast cells lacking Mre11 nuclease activity. Foldback priming at DNA double-strand breaks (DSBs) is one mechanism proposed for the generation of inverted duplications. However, the other pathway steps have not been fully elucidated. Here, we show that a DSB induced near natural inverted repeats drives high frequency inverted duplication in Sae2 and Mre11-deficient cells. We find that DNA polymerase δ proof-reading activity, but not Rad1 nuclease, trims the heterologous flaps formed after foldback annealing. Additionally, Pol32 is required for the generation of inverted duplications, suggesting that Pol δ catalyzes fill-in synthesis primed from the foldback to create a hairpin-capped chromosome that is subsequently replicated to form a dicentric inversion chromosome. Finally, we show that stabilization of the dicentric chromosome after breakage involves telomere capture by non-reciprocal translocation mediated by repeat sequences or by deletion of one centromere.

Rare germline variants in POLE and POLD1 encoding the catalytic subunits of DNA polymerases ε and δ in glioma families.

Pathogenic germline variants in the DNA polymerase genes POLE and POLD1 cause polymerase proofreading-associated polyposis, a dominantly inherited disorder with increased risk of colorectal carcinomas and other tumors. POLE/POLD1 variants may result in high somatic mutation and neoantigen loads that confer susceptibility to immune checkpoint inhibitors (ICIs). To explore the role of POLE/POLD1 germline variants in glioma predisposition, whole-exome sequencing was applied to leukocyte DNA of glioma patients from 61 tumor families with at least one glioma case each. Rare heterozygous POLE/POLD1 missense variants predicted to be deleterious were identified in glioma patients from 10 (16%) families, co-segregating with the tumor phenotype in families with available DNA from several tumor patients. Glioblastoma patients carrying rare POLE variants had a mean overall survival of 21 months. Additionally, germline variants in POLD1, located at 19q13.33, were detected in 2/34 (6%) patients with 1p/19q-codeleted oligodendrogliomas, while POLE variants were identified in 2/4 (50%) glioblastoma patients with a spinal metastasis. In 13/15 (87%) gliomas from patients carrying POLE/POLD1 variants, features of defective polymerase proofreading, e.g. hypermutation, POLE/POLD1-associated mutational signatures, multinucleated cells, and increased intratumoral T cell response, were observed. In a CRISPR/Cas9-derived POLE-deficient LN-229 glioblastoma cell clone, a mutator phenotype and delayed S phase progression were detected compared to wildtype POLE cells. Our data provide evidence that rare POLE/POLD1 germline variants predispose to gliomas that may be susceptible to ICIs. Data compiled here suggest that glioma patients carrying POLE/POLD1 variants may be recognized by cutaneous manifestations, e.g. café-au-lait macules, and benefit from surveillance colonoscopy.

Recommendations for the classification of germline variants in the exonuclease domain of POLE and POLD1.

BACKGROUND: Germline variants affecting the proofreading activity of polymerases epsilon and delta cause a hereditary cancer and adenomatous polyposis syndrome characterized by tumors with a high mutational burden and a specific mutational spectrum. In addition to the implementation of multiple pieces of evidence for the classification of gene variants, POLE and POLD1 variant classification is particularly challenging given that non-disruptive variants affecting the proofreading activity of the corresponding polymerase are the ones associated with cancer. In response to an evident need in the field, we have developed gene-specific variant classification recommendations, based on the ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology) criteria, for the assessment of non-disruptive variants located in the sequence coding for the exonuclease domain of the polymerases. METHODS: A training set of 23 variants considered pathogenic or benign was used to define the usability and strength of the ACMG/AMP criteria. Population frequencies, computational predictions, co-segregation data, phenotypic and tumor data, and functional results, among other features, were considered. RESULTS: Gene-specific variant classification recommendations for non-disruptive variants located in the exonuclease domain of POLE and POLD1 were defined. The resulting recommendations were applied to 128 exonuclease domain variants reported in the literature and/or public databases. A total of 17 variants were classified as pathogenic or likely pathogenic, and 17 as benign or likely benign. CONCLUSIONS: Our recommendations, with room for improvement in the coming years as more information become available on carrier families, tumor molecular characteristics and functional assays, are intended to serve the clinical and scientific communities and help improve diagnostic performance, avoiding variant misclassifications.

A four-point molecular handover during Okazaki maturation.

DNA replication introduces thousands of RNA primers into the lagging strand that need to be removed for replication to be completed. In Escherichia coli when the replicative DNA polymerase Pol IIIα terminates at a previously synthesized RNA primer, DNA Pol I takes over and continues DNA synthesis while displacing the downstream RNA primer. The displaced primer is subsequently excised by an endonuclease, followed by the sealing of the nick by a DNA ligase. Yet how the sequential actions of Pol IIIα, Pol I polymerase, Pol I endonuclease and DNA ligase are coordinated is poorly defined. Here we show that each enzymatic activity prepares the DNA substrate for the next activity, creating an efficient four-point molecular handover. The cryogenic-electron microscopy structure of Pol I bound to a DNA substrate with both an upstream and downstream primer reveals how it displaces the primer in a manner analogous to the monomeric helicases. Moreover, we find that in addition to its flap-directed nuclease activity, the endonuclease domain of Pol I also specifically cuts at the RNA-DNA junction, thus marking the end of the RNA primer and creating a 5' end that is a suitable substrate for the ligase activity of LigA once all RNA has been removed.

Engineering of the DNA replication and repair machinery to develop binary mutators for rapid genome evolution of Corynebacterium glutamicum.

Corynebacterium glutamicum is an important industrial workhorse for production of amino acids and chemicals. Although recently developed genome editing technologies have advanced the rational genetic engineering of C. glutamicum, continuous genome evolution based on genetic mutators is still unavailable. To address this issue, the DNA replication and repair machinery of C. glutamicum was targeted in this study. DnaQ, the homolog of ϵ subunit of DNA polymerase III responsible for proofreading in Escherichia coli, was proven irrelevant to DNA replication fidelity in C. glutamicum. However, the histidinol phosphatase (PHP) domain of DnaE1, the α subunit of DNA polymerase III, was characterized as the key proofreading element and certain variants with PHP mutations allowed elevated spontaneous mutagenesis. Repression of the NucS-mediated post-replicative mismatch repair pathway or overexpression of newly screened NucS variants also impaired the DNA replication fidelity. Simultaneous interference with the DNA replication and repair machinery generated a binary genetic mutator capable of increasing the mutation rate by up to 2352-fold. The mutators facilitated rapid evolutionary engineering of C. glutamicum to acquire stress tolerance and protein overproduction phenotypes. This study provides efficient tools for evolutionary engineering of C. glutamicum and could inspire the development of mutagenesis strategy for other microbial hosts.

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

BACKGROUND: 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). METHODS: 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. RESULTS: 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). CONCLUSION: 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.

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.