GWAS loci associated with Chagas cardiomyopathy influences DNA methylation levels.

A recent genome-wide association study (GWAS) identified a locus in chromosome 11 associated with the chronic cardiac form of Chagas disease. Here we aimed to elucidate the potential functional mechanism underlying this genetic association by analyzing the correlation among single nucleotide polymorphisms (SNPs) and DNA methylation (DNAm) levels as cis methylation quantitative trait loci (cis-mQTL) within this region. A total of 2,611 SNPs were tested against 2,647 DNAm sites, in a subset of 37 chronic Chagas cardiomyopathy patients and 20 asymptomatic individuals from the GWAS. We identified 6,958 significant cis-mQTLs (False Discovery Rate [FDR]<0.05) at 1 Mb each side of the GWAS leading variant, where six of them potentially modulate the expression of the SAC3D1 gene, the reported gene in the previous GWAS. In addition, a total of 268 cis-mQTLs showed differential methylation between chronic Chagas cardiomyopathy patients and asymptomatic individuals. The most significant cis-mQTLs mapped in the gene bodies of POLA2 (FDR = 1.04x10-11), PLAAT3 (FDR = 7.22x10-03), and CCDC88B (FDR = 1.89x10-02) that have been associated with cardiovascular and hematological traits in previous studies. One of the most relevant interactions correlated with hypermethylation of CCDC88B. This gene is involved in the inflammatory response, and its methylation and expression levels have been previously reported in Chagas cardiomyopathy. Our findings support the functional relevance of the previously associated genomic region, highlighting the regulation of novel genes that could play a role in the chronic cardiac form of the disease.

Small tandem DNA duplications result from CST-guided Pol α-primase action at DNA break termini.

Small tandem duplications of DNA occur frequently in the human genome and are implicated in the aetiology of certain human cancers. Recent studies have suggested that DNA double-strand breaks are causal to this mutational class, but the underlying mechanism remains elusive. Here, we identify a crucial role for DNA polymerase α (Pol α)-primase in tandem duplication formation at breaks having complementary 3' ssDNA protrusions. By including so-called primase deserts in CRISPR/Cas9-induced DNA break configurations, we reveal that fill-in synthesis preferentially starts at the 3' tip, and find this activity to be dependent on 53BP1, and the CTC1-STN1-TEN1 (CST) and Shieldin complexes. This axis generates near-blunt ends specifically at DNA breaks with 3' overhangs, which are subsequently repaired by non-homologous end-joining. Our study provides a mechanistic explanation for a mutational signature abundantly observed in the genomes of species and cancer cells.

Studies of multifunctional DNA polymerase I from the extremely radiation resistant Deinococcus radiodurans: Recombinant expression, purification and characterization of the full-length protein and its large fragment.

Deinococcus radiodurans is a bacterium with extreme resistance to desiccation and radiation. Although the origins of this extreme resistance have not been fully elucidated, an efficient DNA repair machinery that includes the enzyme DNA polymerase I, is potentially crucial as part of a protection mechanism. Here we have cloned and performed small, medium, and large-scale expression of full-length D. radiodurans DNA polymerase I (DrPolI) as well as the large/Klenow fragment (DrKlenow). We then carried out functional characterization of 5' exonuclease, DNA strand displacement and polymerase activities of these proteins using gel-based and molecular beacon-based biochemical assays. With the same expression and purification strategy, we got higher yield in the production of DrKlenow than of the full-length protein, approximately 2.5 mg per liter of culture. Moreover, we detected a prominent 5' exonuclease activity of DrPolI in vitro. This activity and, DrKlenow strand displacement and DNA polymerase activities are preferentially stimulated at pH 8.0-8.5 and are reduced by addition of NaCl. Interestingly, both protein variants are more thermostable at pH 6.0-6.5. The characterization of DrPolI's multiple functions provides new insights into the enzyme's role in DNA repair pathways, and how the modulation of these functions is potentially used by D. radiodurans as a survival strategy.

Pol α-primase dependent nuclear localization of the mammalian CST complex.

The human CST complex composed of CTC1, STN1, and TEN1 is critically involved in telomere maintenance and homeostasis. Specifically, CST terminates telomere extension by inhibiting telomerase access to the telomeric overhang and facilitates lagging strand fill in by recruiting DNA Polymerase alpha primase (Pol α-primase) to the telomeric C-strand. Here we reveal that CST has a dynamic intracellular localization that is cell cycle dependent. We report an increase in nuclear CST several hours after the initiation of DNA replication, followed by exit from the nucleus prior to mitosis. We identify amino acids of CTC1 involved in Pol α-primase binding and nuclear localization. We conclude, the CST complex does not contain a nuclear localization signal (NLS) and suggest that its nuclear localization is reliant on Pol α-primase. Hypomorphic mutations affecting CST nuclear import are associated with telomere syndromes and cancer, emphasizing the important role of this process in health.

A multifunctional DNA polymerase I involves in the maturation of Okazaki fragments during the lagging-strand DNA synthesis in Helicobacter pylori.

Helicobacter pylori is the most infectious human pathogen that causes gastritis, peptic ulcers and stomach cancer. H. pylori DNA polymerase I (HpPol I) is found to be essential for the viability of H. pylori, but its intrinsic property and attribution to the H. pylori DNA replication remain unclear. HpPol I contains a 5'→3' exonuclease (5'-Exo) and DNA polymerase (Pol) domain, respectively, but lacks a 3'→5' exonuclease, or error proofreading activity. In this study, we characterized the 5'-Exo and Pol functions of HpPol I and found that HpPol I is a multifunctional protein displaying DNA nick translation, strand-displacement synthesis, RNase H-like, structure-specific endonuclease and exonuclease activities. In the in vitro DNA replication assay, we further demonstrated that the 5'-Exo and Pol domains of HpPol I can cooperate to fill in the DNA gap, remove the unwanted RNA primer from a RNA/DNA hybrid and create a ligatable nick for the DNA ligase A of H. pylori to restore the normal duplex DNA. Altogether, our study suggests that the two catalytic domains of HpPol I may synergistically play an important role in the maturation of Okazaki fragments during the lagging-strand DNA synthesis in H. pylori. Like the functions of DNA polymerase I in Escherichia coli, HpPol I may involve in both DNA replication and repair in H. pylori.

Involvement of POLA2 in Double Strand Break Repair and Genotoxic Stress.

Cellular survival is dependent on the efficient replication and transmission of genomic information. DNA damage can be introduced into the genome by several different methods, one being the act of DNA replication. Replication is a potent source of DNA damage and genomic instability, especially through the formation of DNA double strand breaks (DSBs). DNA polymerase alpha is responsible for replication initiation. One subunit of the DNA polymerase alpha replication machinery is POLA2. Given the connection between replication and genomic instability, we decided to examine the role of POLA2 in DSB repair, as little is known about this topic. We found that loss of POLA2 leads to an increase in spontaneous DSB formation. Loss of POLA2 also slows DSB repair kinetics after treatment with etoposide and inhibits both of the major double strand break repair pathways: non-homologous end-joining and homologous recombination. In addition, loss of POLA2 leads to increased sensitivity to ionizing radiation and PARP1 inhibition. Lastly, POLA2 expression is elevated in glioblastoma multiforme tumors and correlates with poor overall patient survival. These data demonstrate a role for POLA2 in DSB repair and resistance to genotoxic stress.

CircRNA circ_POLA2 promotes lung cancer cell stemness via regulating the miR-326/GNB1 axis.

Circular RNAs (CircRNAs) are a group of noncoding RNAs that have essential function in the development and progression of various cancers. The expression pattern and function of circRNA in lung cancer is not fully understood. In the present study, we aimed to investigate the expression profiles and underlying mechanism of circRNA circ_POLA2 in lung cancer cell stemness. Circ_POLA2 was highly expressed in lung cancer tissues and predicted a poor prognosis in lung cancer patients. Knockdown of circ_POLA2 inhibited the stemness of lung cancer cells, which is evident by the decreased sphere-formation ability, ALDH1 activity, and stemness marker expression, but had no effects on cell viability. Mechanistically, circ_POLA2 functioned as a ceRNA by sponging miR-326. Furthermore, miR-326 negatively regulated G protein subunit beta 1 (GNB1) expression by targeting its 3'-UTR (untranslated region). Intriguingly, we found that GNB1 was overexpressed and associated with poor prognosis in lung cancer patients. Overexpression of GNB1 could antagonize the inhibitory effect of circ_POLA2 knockdown on lung cancer cell stemness. In conclusion, circ_POLA2 promotes lung cancer cell stemness and progression via regulating the miR-326/GNB1 axis, which might serve as a novel therapeutic target for lung cancer patients.

CHK1 Inhibition Is Synthetically Lethal with Loss of B-Family DNA Polymerase Function in Human Lung and Colorectal Cancer Cells.

Checkpoint kinase 1 (CHK1) is a key mediator of the DNA damage response that regulates cell-cycle progression, DNA damage repair, and DNA replication. Small-molecule CHK1 inhibitors sensitize cancer cells to genotoxic agents and have shown single-agent preclinical activity in cancers with high levels of replication stress. However, the underlying genetic determinants of CHK1 inhibitor sensitivity remain unclear. We used the developmental clinical drug SRA737 in an unbiased large-scale siRNA screen to identify novel mediators of CHK1 inhibitor sensitivity and uncover potential combination therapies and biomarkers for patient selection. We identified subunits of the B-family of DNA polymerases (POLA1, POLE, and POLE2) whose silencing sensitized the human A549 non-small cell lung cancer (NSCLC) and SW620 colorectal cancer cell lines to SRA737. B-family polymerases were validated using multiple siRNAs in a panel of NSCLC and colorectal cancer cell lines. Replication stress, DNA damage, and apoptosis were increased in human cancer cells following depletion of the B-family DNA polymerases combined with SRA737 treatment. Moreover, pharmacologic blockade of B-family DNA polymerases using aphidicolin or CD437 combined with CHK1 inhibitors led to synergistic inhibition of cancer cell proliferation. Furthermore, low levels of POLA1, POLE, and POLE2 protein expression in NSCLC and colorectal cancer cells correlated with single-agent CHK1 inhibitor sensitivity and may constitute biomarkers of this phenotype. These findings provide a potential basis for combining CHK1 and B-family polymerase inhibitors in cancer therapy. SIGNIFICANCE: These findings demonstrate how the therapeutic benefit of CHK1 inhibitors may potentially be enhanced and could have implications for patient selection and future development of new combination therapies.

Defective DNA Polymerase α-Primase Leads to X-Linked Intellectual Disability Associated with Severe Growth Retardation, Microcephaly, and Hypogonadism.

Replicating the human genome efficiently and accurately is a daunting challenge involving the duplication of upward of three billion base pairs. At the core of the complex machinery that achieves this task are three members of the B family of DNA polymerases: DNA polymerases α, δ, and ε. Collectively these multimeric polymerases ensure DNA replication proceeds at optimal rates approaching 2 × 103 nucleotides/min with an error rate of less than one per million nucleotides polymerized. The majority of DNA replication of undamaged DNA is conducted by DNA polymerases δ and ε. The DNA polymerase α-primase complex performs limited synthesis to initiate the replication process, along with Okazaki-fragment synthesis on the discontinuous lagging strand. An increasing number of human disorders caused by defects in different components of the DNA-replication apparatus have been described to date. These are clinically diverse and involve a wide range of features, including variable combinations of growth delay, immunodeficiency, endocrine insufficiencies, lipodystrophy, and cancer predisposition. Here, by using various complementary approaches, including classical linkage analysis, targeted next-generation sequencing, and whole-exome sequencing, we describe distinct missense and splice-impacting mutations in POLA1 in five unrelated families presenting with an X-linked syndrome involving intellectual disability, proportionate short stature, microcephaly, and hypogonadism. POLA1 encodes the p180 catalytic subunit of DNA polymerase α-primase. A range of replicative impairments could be demonstrated in lymphoblastoid cell lines derived from affected individuals. Our findings describe the presentation of pathogenic mutations in a catalytic component of a B family DNA polymerase member, DNA polymerase α.

DNA polymerase η contributes to genome-wide lagging strand synthesis.

DNA polymerase η (pol η) is best known for its ability to bypass UV-induced thymine-thymine (T-T) dimers and other bulky DNA lesions, but pol η also has other cellular roles. Here, we present evidence that pol η competes with DNA polymerases α and δ for the synthesis of the lagging strand genome-wide, where it also shows a preference for T-T in the DNA template. Moreover, we found that the C-terminus of pol η, which contains a PCNA-Interacting Protein motif is required for pol η to function in lagging strand synthesis. Finally, we provide evidence that a pol η dependent signature is also found to be lagging strand specific in patients with skin cancer. Taken together, these findings provide insight into the physiological role of DNA synthesis by pol η and have implications for our understanding of how our genome is replicated to avoid mutagenesis, genome instability and cancer.

DNArCdb: A database of cancer biomarkers in DNA repair genes that includes variants related to multiple cancer phenotypes.

Functioning DNA repair capabilities are vital for organisms to ensure that the biological information is preserved and correctly propagated. Disruptions in DNA repair pathways can result in the accumulation of DNA mutations, which may lead to onset of complex disease such as cancer. The discovery and characterization of cancer-related biomarkers may allow early diagnosis and targeted treatment, which could significantly contribute to the survival rates of cancer patients. To this end, we have applied a hypothesis driven bioinformatics approach to identify biomarkers related to 25 different DNA repair enzymes, in combination with structural analysis of six selected missense mutations of newly discovered SNPs that are associated with cancer phenotypes. Our search on 8 distinct cancer databases uncovered 43 missense SNPs that statistically significantly associated at least one phenotype. Moreover, nine of these missense SNPs are statistically significantly associated with two or more cancers. In addition, we have performed classical molecular dynamics to characterize the impact of rs10018786 on POLN, which results in the M310 L Pol ν variant, and rs3218784 on POLI, which results in the I236 M Pol ι. Our results suggest that both of these cancer-associated variants result in noticeable structural and dynamical changes compared with their respective wild-type proteins.

CTC1-STN1 terminates telomerase while STN1-TEN1 enables C-strand synthesis during telomere replication in colon cancer cells.

Telomerase elongates the telomeric G-strand to prevent telomere shortening through conventional DNA replication. However, synthesis of the complementary C-strand by DNA polymerase α is also required to maintain telomere length. Polymerase α cannot perform this role without the ssDNA binding complex CST (CTC1-STN1-TEN1). Here we describe the roles of individual CST subunits in telomerase regulation and G-overhang maturation in human colon cancer cells. We show that CTC1-STN1 limits telomerase action to prevent G-overhang overextension. CTC1-/- cells exhibit telomeric DNA damage and growth arrest due to overhang elongation whereas TEN1-/- cells do not. However, TEN1 is essential for C-strand synthesis and TEN1-/- cells exhibit progressive telomere shortening. DNA binding analysis indicates that CTC1-STN1 retains affinity for ssDNA but TEN1 stabilizes binding. We propose CTC1-STN1 binding is sufficient to terminate telomerase action but altered DNA binding dynamics renders CTC1-STN1 unable to properly engage polymerase α on the overhang for C-strand synthesis.

Subunits of the DNA polymerase alpha-primase complex promote Notch-mediated proliferation with discrete and shared functions in C. elegans germline.

Notch receptor signaling is a highly conserved cell communication system in most multicellular organisms and plays a critical role at several junctures in animal development. In Caenorhabditis elegans,GLP-1/Notch signaling is essential for both germline stem cell maintenance and germ cell proliferation during gonad development. Here, we show that subunits (POLA-1, DIV-1, PRI-1, and PRI-2) of the DNA polymerase alpha-primase complex are required for germ cell proliferation in response to GLP-1/Notch signaling in different tissues at different developmental stages. Specifically, genetic and functional analyses demonstrated that (a) maternally contributed DIV-1 (regulatory subunit) is indispensable non-cell autonomously for GLP-1/Notch-mediated germ cell proliferation during early larval development, whereas POLA-1 (catalytic subunit) and two primase subunits, PRI-1 and PRI-2, do not appear to be essential; (b) germline POLA-1, PRI-1, and PRI-2 play a crucial role in GLP-1/Notch-mediated maintenance of proliferative cell fate during adulthood, while DIV-1 is dispensable; and (c) germline POLA-1, DIV-1, PRI-1, and PRI-2 function in tandem with PUF (Pumilio/FBF) RNA-binding proteins to maintain germline stem cells in the adult gonad. These findings suggest that the subunits of the DNA polymerase alpha-primase complex exhibit both discrete and shared functions in GLP-1/Notch or PUF-mediated germ cell dynamics in C. elegans. These findings link the biological functions of DNA replication machineries to signals that maintain a stem cell population, and may have further implications for Notch-dependent tumors.

Induction of Apoptosis in Metastatic Breast Cancer Cells: XV. Downregulation of DNA Polymerase-α - Helicase Complex (Replisomes) and Glyco-Genes.

In normal and cancer cells, successful cell division requires accurate duplication of chromosomal DNA. All cells require a multiprotein DNA duplication system (replisomes) for their existence. However, death of normal cells in our body occurs through the apoptotic process. During apoptotic process several crucial genes are downregulated with the upregulation of caspase pathways, leading to ultimate degradation of genomic DNA. In metastatic cancer cells (SKBR-3, MCF -7, and MDA-462), this process is inhibited to achieve immortality as well as overexpression of the enzymes for the synthesis of marker molecules. It is believed that the GSL of the lacto family such as LeX, SA-LeX, LeY, Lea, and Leb are markers on the human colon and breast cancer cells. Recently, we have characterized that a few apoptotic chemicals (cis-platin, L-PPMP, D-PDMP, GD3 ganglioside, GD1b ganglioside, betulinic acid, tamoxifen, and melphalan) in low doses kill metastatic breast cancer cells. The apoptosis-inducing agent (e.g., cis-platin) showed inhibition of DNA polymerase/helicase (part of the replisomes) and also modulated (positively) a few glycolipid-glycosyltransferase (GSL-GLTs) transcriptions in the early stages (within 2 h after treatment) of apoptosis. These Lc-family GSLs are also present on the surfaces of human breast and colon carcinoma cells. It is advantageous to deliver these apoptotic chemicals through the metastatic cell surfaces containing high concentration of marker glycolipids (Lc-GSLs). Targeted application of apoptotic chemicals (in micro scale) to kill the cancer cells would be an ideal way to inhibit the metastatic growth of both breast and colon cancer cells. It was observed in three different breast cancer lines (SKBR-3, MDA-468, and MCF-7) that in 2 h very little apoptotic process had started, but predominant biochemical changes (including inactivation of replisomes) started between 6 and 24 h of the drug treatments. The contents of replisomes (replisomal complexes) during induction of apoptosis are not known. It is known that DNA helicase activities (major proteins catalyze the melting of dsDNA strands) change during apoptotic induction process. Previously DNA Helicase-III was characterized as a component of the replication complexes isolated from carcinoma cells and normal rapid growing embryonic chicken brain cells. Helicase activities were assayed by a novel method (combined immunoprecipitation-ROME assay), and DNA polymerase-alpha activities were determined by regular chain extension of nicked "ACT-DNA," by determining values obtained from +/- aphidicolin added to the incubation mixtures. Very little is known about the stability of the "replication complexes" (or replisomes) during the apoptotic process. DNA helicases are motor proteins that catalyze the melting of genomic DNA during replication, repair, and recombination processes. In all three breast carcinoma cell lines (SKBR-3, MCF-7, and MDA-468), a common trend, decrease of activities of DNA polymerase-alpha and Helicase-III (estimated and detected with a polyclonal antibody), was observed, after cis-platin- and L-PPMP-induced apoptosis. Previously our laboratory has documented downregulation (within 24-48 h) of several GSL-GLTs with these apoptotic reagents in breast and colon cancer cells also. Perhaps induced apoptosis would improve the prognosis in metastatic breast and colon cancer patients.

Chromatin Controls DNA Replication Origin Selection, Lagging-Strand Synthesis, and Replication Fork Rates.

The integrity of eukaryotic genomes requires rapid and regulated chromatin replication. How this is accomplished is still poorly understood. Using purified yeast replication proteins and fully chromatinized templates, we have reconstituted this process in vitro. We show that chromatin enforces DNA replication origin specificity by preventing non-specific MCM helicase loading. Helicase activation occurs efficiently in the context of chromatin, but subsequent replisome progression requires the histone chaperone FACT (facilitates chromatin transcription). The FACT-associated Nhp6 protein, the nucleosome remodelers INO80 or ISW1A, and the lysine acetyltransferases Gcn5 and Esa1 each contribute separately to maximum DNA synthesis rates. Chromatin promotes the regular priming of lagging-strand DNA synthesis by facilitating DNA polymerase α function at replication forks. Finally, nucleosomes disrupted during replication are efficiently re-assembled into regular arrays on nascent DNA. Our work defines the minimum requirements for chromatin replication in vitro and shows how multiple chromatin factors might modulate replication fork rates in vivo.

Optimal numbers of residues in linkers of DNA polymerase I, T7 primase and DNA polymerase IV.

DNA polymerase I (PolI), T7 primase and DNA polymerase IV (Dpo4) have a common feature in their structures that the two main domains are connected by an unstructured polypeptide linker. To perform their specific enzymatic activities, the enzymes are required to rearrange the position and orientation of one domain relative to the other into an active mode. Here, we show that the three enzymes share the same mechanism of the transition from the inert to active modes and use the minimum numbers of residues in their linkers to achieve the most efficient transitions. The transition time to the finally active mode is sensitively dependent on the stretched length of the linker in the finally active mode while is insensitive to the position and orientation in the initially inert state. Moreover, we find that for any enzyme whose two domains are connected by an unstructured flexible linker, the stretched length (L) of the linker in the finally active mode and the optimal number (Nopt) of the residues in the linker satisfy relation L ≈ αNopt, with α = 0.24-0.27 nm being a constant insensitive to the system.

[Surgical aspects of indications and techniques for adenomatous polyposis variants]. / Chirurgische Aspekte zu Indikation und Technik bei Varianten der adenomatösen Polyposis.

Due to the advances in molecular genetic diagnostics of adenomatous polyposis variants, identification of patients with a genetic predisposition and their at risk relatives is becoming increasingly important in clinical practice. Precise knowledge of the specific risk profile is gaining significance especially for surgeons and requires a clinically differentiated approach in order to correctly identify the indications for prophylactic surgery. In this article reference will be made to the technical details of the pouch operation rather than the decision-making process per se, since this has become common knowledge for specialized colorectal surgeons. Besides the more commonly known polyposis syndromes, such as familial adenomatous polyposis (FAP), surgeons should nowadays at least be able to clinically distinguish between attenuated and classical variants of FAP, be aware of MUTYH-associated polyposis (MAP) and also the new polyposis syndrome polymerase proofreading-associated polyposis (PPAP). Surgeons should be familiar with the specific indications and extent of surgery for prophylactic organ removal in the lower gastrointestinal tract in order to be able to competently advise patients.

The antitumor toxin CD437 is a direct inhibitor of DNA polymerase α.

CD437 is a retinoid-like small molecule that selectively induces apoptosis in cancer cells, but not in normal cells, through an unknown mechanism. We used a forward-genetic strategy to discover mutations in POLA1 that coincide with CD437 resistance (POLA1(R)). Introduction of one of these mutations into cancer cells by CRISPR-Cas9 genome editing conferred CD437 resistance, demonstrating causality. POLA1 encodes DNA polymerase α, the enzyme responsible for initiating DNA synthesis during the S phase of the cell cycle. CD437 inhibits DNA replication in cells and recombinant POLA1 activity in vitro. Both effects are abrogated by the identified POLA1 mutations, supporting POLA1 as the direct antitumor target of CD437. In addition, we detected an increase in the total fluorescence intensity and anisotropy of CD437 in the presence of increasing concentrations of POLA1 that is consistent with a direct binding interaction. The discovery of POLA1 as the direct anticancer target for CD437 has the potential to catalyze the development of CD437 into an anticancer therapeutic.

Knockdown of POLA2 increases gemcitabine resistance in lung cancer cells.

BACKGROUND: Gemcitabine is used as a standard drug treatment for non-small cell lung cancer (NSCLC), but treatment responses vary among patients. Our previous studies demonstrated that POLA2 + 1747 GG/GA single nucleotide polymorphism (SNP) improves differential survivability and mortality in NSCLC patients. Here, we determined the association between POLA2 and gemcitabine treatment in human lung cancer cells. RESULTS: Human PC9, H1299 and H1650 lung cancer cell lines were treated with 0.01-100 µM gemcitabine for 72 h. Although all 3 cell lines showed decreased cell viability upon gemcitabine treatment, H1299 was found to be the most sensitive to gemcitabine treatment. Next, sequencing was performed to determine if POLA2 + 1747 SNP might be involved in gemcitabine sensitivity. Data revealed that all 3 cell lines harbored the wild-type POLA2 + 1747 GG SNP, indicating that the POLA2 + 1747 SNP might not be responsible for gemcitabine sensitivity in the cell lines studied. Silencing of POLA2 gene in H1299 was then carried out by siRNA transfection, followed by gemcitabine treatment to determine the effect of POLA2 knockdown on chemosensitivity to gemcitabine. Results showed that H1299 exhibited increased resistance to gemcitabine after POLA2 knockdown, suggesting that POLA2 does not act alone and may cooperate with other interacting partners to cause gemcitabine resistance. CONCLUSIONS: Collectively, our findings showed that knockdown of POLA2 increases gemcitabine resistance in human lung cancer cells. We propose that POLA2 may play a role in gemcitabine sensitivity and can be used as a prognostic biomarker of patient outcome in NSCLC pathogenesis.