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.

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.

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.

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.

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 6 months. He manifested progressive neurological regression and ultimately died at age 4 years. 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.

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.

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.

Stepwise requirements for polymerases δ and θ in theta-mediated end joining.

Timely repair of chromosomal double-strand breaks is required for genome integrity and cellular viability. The polymerase theta-mediated end joining pathway has an important role in resolving these breaks and is essential in cancers defective in other DNA repair pathways, thus making it an emerging therapeutic target1. It requires annealing of 2-6 nucleotides of complementary sequence, microhomologies, that are adjacent to the broken ends, followed by initiation of end-bridging DNA synthesis by polymerase θ. However, the other pathway steps remain inadequately defined, and the enzymes required for them are unknown. Here we demonstrate requirements for exonucleolytic digestion of unpaired 3' tails before polymerase θ can initiate synthesis, then a switch to a more accurate, processive and strand-displacing polymerase to complete repair. We show the replicative polymerase, polymerase δ, is required for both steps; its 3' to 5' exonuclease activity for flap trimming, then its polymerase activity for extension and completion of repair. The enzymatic steps that are essential and specific to this pathway are mediated by two separate, sequential engagements of the two polymerases. The requisite coupling of these steps together is likely to be facilitated by physical association of the two polymerases. This pairing of polymerase δ with a polymerase capable of end-bridging synthesis, polymerase θ, may help to explain why the normally high-fidelity polymerase δ participates in genome destabilizing processes such as mitotic DNA synthesis2 and microhomology-mediated break-induced replication3.

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.

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.

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.

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.

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 POLD1 promotes proliferation and metastasis of bladder cancer by stabilizing MYC.

To date, most studies on the DNA polymerase, POLD1, have focused on the effect of POLD1 inactivation mutations in tumors. However, the implications of high POLD1 expression in tumorigenesis remains elusive. Here, we determine that POLD1 has a pro-carcinogenic role in bladder cancer (BLCA) and is associated to the malignancy and prognosis of BLCA. Our studies demonstrate that POLD1 promotes the proliferation and metastasis of BLCA via MYC. Mechanistically, POLD1 stabilizes MYC in a manner independent of its' DNA polymerase activity. Instead, POLD1 attenuates FBXW7-mediated ubiquitination degradation of MYC by directly binding to the MYC homology box 1 domain competitively with FBXW7. Moreover, we find that POLD1 forms a complex with MYC to promote the transcriptional activity of MYC. In turn, MYC increases expression of POLD1, forming a POLD1-MYC positive feedback loop to enhance the pro-carcinogenic effect of POLD1-MYC on BLCA. Overall, our study identifies POLD1 as a promotor of BCLA via a MYC driven mechanism and suggest its potential as biomarker for BLCA.

PARP1 and POLD2 as prognostic biomarkers for multiple myeloma in autologous stem cell transplant.

Multiple Myeloma (MM) is an incurable plasma cell malignancy often treated by autologous stem cell transplant (ASCT). Clinical response to ASCT has been associated with DNA repair efficiency. Here we interrogated the role of the base excision DNA repair (BER) pathway in MM response to ASCT. Across 450 clinical samples and six disease stages, expression levels of genes in the BER pathway were found to be highly upregulated during the development of MM. In a separate cohort of 559 patients with MM treated with ASCT, expression of BER pathway members MPG and PARP3 was positively associated with overall survival (OS) while expression of PARP1, POLD1, and POLD2 was negatively associated with OS. In a validation cohort of 356 patients with MM treated with ASCT, PARP1 and POLD2 findings were replicated. In patients with MM who never received ASCT (n=319), PARP1 and POLD2 were not associated with OS, suggesting that the prognostic effect of these genes may be treatment-dependent. In preclinical models of MM, synergy was observed in anti-tumor activity when poly (ADPribose) polymerase (PARP) inhibitors (olaparib, talazoparib) were used in combination with melphalan. The negative prognosis associated with PARP1 and POLD2 expression along with the apparent melphalan-sensitizing effect of PARP inhibition may suggest this pathway as a potential biomarker in patients with MM in the setting of ASCT. Further understanding of the role of the BER pathway in MM is vital to improve therapeutic strategies related to ASCT.

A novel role for Mms2 in the control of spontaneous mutagenesis and Pol3 abundance.

Mms2 is a ubiquitin E2-variant protein with a very well-documented function in the tolerance pathway that protects both human and yeast cells from the lethal and mutagenic effects of DNA damage. Interestingly, a high expression level of human MMS2 is associated with poor survival prognosis in different cancer diseases. Here we have analyzed the physiological effects of Mms2 overproduction in yeast cells. We show that an increased level of this protein causes a spontaneous mutator effect independent of Ubc13, a cognate partner of Mms2 in the PCNA-polyubiquitinating complex responsible for the template switch. Instead, this new promutagenic role of Mms2 requires Ubc4 (E2) and two ubiquitin ligases of HECT and RING families, Rsp5 and Not4, respectively. We have established that the promutagenic activity of Mms2 is dependent on the activities of error-prone DNA polymerase ζ and Rev1. Additionally, it requires the ubiquitination of K164 in PCNA which facilitates recruitment of these translesion polymerases to the replication complex. Importantly, we have established also that the cellular abundance of Mms2 influences the cellular level of Pol3, the catalytic subunit of replicative DNA polymerase δ. Lack of Mms2 increases the Pol3 abundance, whereas in response to Mms2 overproduction the Pol3 level decreases. We hypothesize that increased levels of spontaneous mutagenesis may result from the Mms2-induced reduction in Pol3 accumulation leading to increased participation of error-prone polymerase ζ in the replication complex.

From cue to meaning: The involvement of POLD1 gene in DNA replication, repair and aging.

Aging is an extremely complex biological process. Aging, cancer and inflammation represent a trinity, object of many interesting researches. The accumulation of DNA damage and its consequences progressively interfere with cellular function and increase susceptibility to developing aging condition. DNA Polymerase delta (Pol δ), encoded by POLD1 gene (MIM#174761) on 19q13.3, is well implicated in many steps of the replication program and repair. Thanks to its exonuclease and polymerase activities, the enzyme is involved in the regulation of the cell cycle, DNA synthesis, and DNA damage repair processes. Damaging variants within the exonuclease domain predispose to cancers, while those occurring in the polymerase active site cause the autosomal dominant Progeroid Syndrome called MDPL, Mandibular hypoplasia, Deafness and Progeroid features with concomitant Lipodystrophy Since DNA damage represents the main cause of ageing and age-related pathologies, an overview of critical Pol δ activities will allow to better understand the associations between DNA damage and nearly every aspect of the ageing process, helping the researchers to counteract all the ageing-pathologies at the same time.

Characterization of the Escherichia coli XPD/Rad3 iron-sulfur helicase YoaA in complex with the DNA polymerase III clamp loader subunit chi (χ).

Escherichia coli YoaA aids in the resolution of DNA damage that halts DNA synthesis in vivo in conjunction with χ, an accessory subunit of DNA polymerase III. YoaA and χ form a discrete complex separate from the DNA polymerase III holoenzyme, but little is known about how YoaA and χ work together to help the replication fork overcome damage. Although YoaA is predicted to be an iron-sulfur helicase in the XPD/Rad3 helicase family based on sequence analysis, the biochemical activities of YoaA have not been described. Here, we characterize YoaA and show that purified YoaA contains iron. YoaA and χ form a complex that is stable through three chromatographic steps, including gel filtration chromatography. When overexpressed in the absence of χ, YoaA is mostly insoluble. In addition, we show the YoaA-χ complex has DNA-dependent ATPase activity. Our measurement of the YoaA-χ helicase activity illustrates for the first time YoaA-χ translocates on ssDNA in the 5' to 3' direction and requires a 5' single-stranded overhang, or ssDNA gap, for DNA/DNA unwinding. Furthermore, YoaA-χ preferentially unwinds forked duplex DNA that contains both 3' and 5' single-stranded overhangs versus duplex DNA with only a 5' overhang. Finally, we demonstrate YoaA-χ can unwind damaged DNA that contains an abasic site or damage on 3' ends that stall replication extension. These results are the first biochemical evidence demonstrating YoaA is a bona fide iron-sulfur helicase, and we further propose the physiologically relevant form of the helicase is YoaA-χ.