Endogenous overexpression of an active phosphorylated form of DNA polymerase ß under oxidative stress in Trypanosoma cruzi.

Trypanosoma cruzi is exposed during its life to exogenous and endogenous oxidative stress, leading to damage of several macromolecules such as DNA. There are many DNA repair pathways in the nucleus and mitochondria (kinetoplast), where specific protein complexes detect and eliminate damage to DNA. One group of these proteins is the DNA polymerases. In particular, Tc DNA polymerase ß participates in kinetoplast DNA replication and repair. However, the mechanisms which control its expression under oxidative stress are still unknown. Here we describe the effect of oxidative stress on the expression and function of Tc DNA polymerase ß To this end parasite cells (epimastigotes and trypomastigotes) were exposed to peroxide during short periods of time. Tc DNA polymerase ß which was associated physically with kinetoplast DNA, showed increased protein levels in response to peroxide damage in both parasite forms analyzed. Two forms of DNA polymerase ß were identified and overexpressed after peroxide treatment. One of them was phosphorylated and active in DNA synthesis after renaturation on polyacrylamide electrophoresis gel. This phosphorylated form showed 3-4-fold increase in both parasite forms. Our findings indicate that these increments in protein levels are not under transcriptional control because the level of Tc DNA polymerase ß mRNA is maintained or slightly decreased during the exposure to oxidative stress. We propose a mechanism where a DNA repair pathway activates a cascade leading to the increment of expression and phosphorylation of Tc DNA polymerase ß in response to oxidative damage, which is discussed in the context of what is known in other trypanosomes which lack transcriptional control.

An oxidized abasic lesion inhibits base excision repair leading to DNA strand breaks in a trinucleotide repeat tract.

Oxidative DNA damage and base excision repair (BER) play important roles in modulating trinucleotide repeat (TNR) instability that is associated with human neurodegenerative diseases and cancer. We have reported that BER of base lesions can lead to TNR instability. However, it is unknown if modifications of the sugar in an abasic lesion modulate TNR instability. In this study, we characterized the effects of the oxidized sugar, 5'-(2-phosphoryl-1,4-dioxobutane)(DOB) in CAG repeat tracts on the activities of key BER enzymes, as well as on repeat instability. We found that DOB crosslinked with DNA polymerase ß and inhibited its synthesis activity in CAG repeat tracts. Surprisingly, we found that DOB also formed crosslinks with DNA ligase I and inhibited its ligation activity, thereby reducing the efficiency of BER. This subsequently resulted in the accumulation of DNA strand breaks in a CAG repeat tract. Our study provides important new insights into the adverse effects of an oxidized abasic lesion on BER and suggests a potential alternate repair pathway through which an oxidized abasic lesion may modulate TNR instability.

Impact of reduced levels of APE1 transcripts on the survival of patients with urothelial carcinoma of the bladder.

Molecular evidence indicates that alterations in genes involved in the maintenance of genome stability may be related to susceptibility to bladder carcinoma. Our goal was to evaluate the prognostic role of base excision repair (BER) genes in a cohort of patients diagnosed with primary urothelial carcinoma of the bladder (UCB). The levels of all APE1, XRCC1 and POLB transcripts were detected by quantitative real-time PCR (qPCR) technique in tumor samples from 52 patients undergoing transurethral resection (TUR) for primary UCB at the Department of Urology, Brazilian National Cancer Institute, Rio de Janeiro. Increased levels of APE1, XRCC1 and POLB transcripts were significantly associated with high-grade tumors when compared to these levels in low-grade tumors (p<0.01) and could be attributed to different mechanisms of transcriptional regulation as a response to tumorigenesis and oxidative stress. By analyzing the collected data in the present study, regardless of pathological grade or stage, univariate analysis revealed that the reduced levels of APE1 transcripts were significantly associated with cancer-specific mortality (p=0.032). Furthermore, the variant genotype (TG/GG) of the APE1 T1349G polymorphism was observed in 75% of a subset of patients who concomitantly experienced reduced levels of the APE1 transcript and death and/or recurrence events. Taken together, our data reinforce the idea that human DNA repair mechanisms must be finely regulated in order to avoid instability leading to tumorigenesis and poor clinical outcomes in UCB patients.

Transient OGG1, APE1, PARP1 and Polß expression in an Alzheimer's disease mouse model.

Alzheimer's disease (AD) is a disease of major public health significance, whose pathogenesis is strongly linked to the presence of fibrillar aggregates of amyloid-beta (Aß) in the aging human brain. We exploited the transgenic (Tg)-ArcSwe mouse model for human AD to explore whether oxidative stress and the capacity to repair oxidative DNA damage via base excision repair (BER) are related to Aß pathology in AD. Tg-ArcSwe mice express variants of Aß, accumulating senile plaques at 4-6 months of age, and develop AD-like neuropathology as adult animals. The relative mRNA levels of genes encoding BER enzymes, including 8-oxoguanine glycosylase (OGG1), AP endonuclease 1 (APE1), polymerase ß (Polß) and poly(ADP-ribose) polymerase 1 (PARP1), were quantified in various brain regions of 6 weeks, 4 months and 12 months old mice. The results show that OGG1 transcriptional expression was higher, and APE1 expression lower, in 4 months old Tg-ArcSwe than in wildtype (wt) mice. Furthermore, Polß transcriptional expression was significantly lower in transgenic 12 months old mice than in wt. Transcriptional profiling also showed that BER repair capacity vary during the lifespan in Tg-ArcSwe and wt mice. The BER expression pattern in Tg-ArcSwe mice thus reflects responses to oxidative stress in vulnerable brain structures.

Involvement of DNA polymerase ß overexpression in the malignant transformation induced by benzo[a]pyrene.

OBJECTIVE: To explore the relationship between DNA polymerase ß (pol ß) overexpression and benzo[a]pyrene (BaP) carcinogenesis. METHODS: Firstly, mouse embryonic fibroblasts that express wild-type level of DNA polymerase ß (pol ß cell) and high level of pol ß (pol ß oe cell) were treated by various concentrations of BaP to determine genetic instability induced by BaP under differential expression levels of pol ß. Secondly, malignant transformation of pol ß cells by low concentration of BaP (20 µM) was determined by soft agar colony formation assay and transformation focus assay. Thirdly, the mRNA and protein levels of BaP-transformed pol ß cells (named pol ß-T cells) was measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot, and the genetic instability of these cells were examined by HPRT gene mutation assay and random amplified polymorphic DNA (RAPD) assay. RESULTS: Pol ß cells were successfully transformed into malignant pol ß-T cells by an exposure to low concentration of BaP for 6 months. Pol ß-T cells exhibited increased levels of pol ß gene expression, HPRT gene mutation frequency and polymorphisms of RAPD products that were comparable to those of pol ß oe cells. CONCLUSION: Pol ß overexpression and its-associated genetic instability may play a key role in BaP carcinogenesis.

A single residue unique to DinB-like proteins limits formation of the polymerase IV multiprotein complex in Escherichia coli.

The activity of DinB is governed by the formation of a multiprotein complex (MPC) with RecA and UmuD. We identified two highly conserved surface residues in DinB, cysteine 66 (C66) and proline 67 (P67). Mapping on the DinB tertiary structure suggests these are noncatalytic, and multiple-sequence alignments indicate that they are unique among DinB-like proteins. To investigate the role of the C66-containing surface in MPC formation, we constructed the dinB(C66A) derivative. We found that DinB(C66A) copurifies with its interacting partners, RecA and UmuD, to a greater extent than DinB. Notably, copurification of RecA with DinB is somewhat enhanced in the absence of UmuD and is further increased for DinB(C66A). In vitro pulldown assays also indicate that DinB(C66A) binds RecA and UmuD better than DinB. We note that the increased affinity of DinB(C66A) for UmuD is RecA dependent. Thus, the C66-containing binding surface appears to be critical to modulate interaction with UmuD, and particularly with RecA. Expression of dinB(C66A) from the chromosome resulted in detectable differences in dinB-dependent lesion bypass fidelity and homologous recombination. Study of this DinB derivative has revealed a key surface on DinB, which appears to modulate the strength of MPC binding, and has suggested a binding order of RecA and UmuD to DinB. These findings will ultimately permit the manipulation of these enzymes to deter bacterial antibiotic resistance acquisition and to gain insights into cancer development in humans.

Two novel determinants of etoposide resistance in small cell lung cancer.

Patient survival in small cell lung cancer (SCLC) is limited by acquired chemoresistance. Here we report the use of a biologically relevant model to identify novel candidate genes mediating in vivo acquired resistance to etoposide. Candidate genes derived from a cDNA microarray analysis were cloned and transiently overexpressed to evaluate their potential functional roles. We identified two promising genes in the DNA repair enzyme DNA polymerase ß and in the neuroendocrine transcription factor NKX2.2. Specific inhibition of DNA polymerase ß reduced the numbers of cells surviving treatment with etoposide and increased the amount of DNA damage in cells. Conversely, stable overexpression of NKX2.2 increased cell survival in response to etoposide in SCLC cell lines. Consistent with these findings, we found that an absence of nuclear staining for NKX2.2 in SCLC primary tumors was an independent predictor of improved outcomes in chemotherapy-treated patients. Taken together, our findings justify future prospective studies to confirm the roles of these molecules in mediating chemotherapy resistance in SCLC.

Links between DNA polymerase beta expression and sensitivity to bleomycin.

Bleomycin (BLM), an important anti-tumor antibiotic, enables cell death through oxidative DNA damage mediated by reactive oxygen species (ROS). However, increasing cellular resistance has become a serious limitation to its clinical application. Base excision repair (BER), the major pathway for repairing oxidative bases, is involved in resistance of DNA-damaging anticancer drugs. DNA polymerase beta (pol ß), a critical BER enzyme, has been reported to play a crucial role in combating BLM-induced oxidative DNA damage, as a result, pol ß inhibition may increase the sensitivity to BLM. To test this hypothesis, we evaluated the sensitivity to BLM using mouse embryo fibroblasts (MEFs) with distinct pol ß expression levels (wild-type, pol ß deficiency) and explored the underlying mechanisms. The results showed that cell viability of pol ß-deficient MEFs was significantly lower than that of isogenic wild type when treated with the same BLM dosage. In addition, increased ROS level, DNA single strand breaks, and chromosomal breakage were observed in pol ß deficient cells, indicating impaired DNA repair and enhanced oxidative DNA damage under pol ß deficiency. In agreement with the findings, an enhanced hprt gene mutation frequency was also detected in pol ß null cells. In summary, this study demonstrated that BLM-induced DNA damage could be repaired through BER pathway and absence of pol ß allows oxidative DNA/chromosome damage and gene mutation, which contributes to BLM hypersensitivity.

[Effects of pol beta on biological characteristics and DNA damage in mouse embryonic fibroblast].

OBJECTIVE: To explore the effect of DNA polymerase beta (pol beta) expression level on biological characteristics of mouse embryonic fibroblast (MEF) and the cellular response to DNA damage induced by potassium dichromate. METHODS: pol beta wild-type cells (pol beta +/+), pol beta null cells (pol beta -/-) and pol beta overexpressed cells (pol beta oe) were applied as a model system. The growth curve of cells was plotted by MTT assay; the doubling time of cells was detected by double time experiment; the spontaneous mutation frequency was determined by HGPRT gene mutation method and single cell gel electrophoresis assay (SCGE) was employed to observe the DNA damage either happened spontaneously or induced by potassium dichromate. RESULTS: Growth characteristic and doubling time of the three kinds of cells were similar and no obvious differences were found on spontaneous DNA damage and mutations frequency among them (P > 0.05). Potassium dichromate increased comet rate and tail length in the three kinds of cells in a concentration dependent way. DNA damage of pol beta -/- cells at the same dosage were more serious than the other cells both in comet rate and tail length (P < 0.05). pol beta oe cells demonstrated more resistant to DNA damage obviously than the others. CONCLUSION: The expression level of pol beta has no significant effect on the biological characteristic and spontaneous mutation frequency of MEF. pol beta knock out cells is more sensitive to DNA damage induced by potassium dichromate, whereas, pol beta over expression can help cells response to DNA damage and protect cells from death in a certain degree.

DNA polymerases beta and lambda bypass thymine glycol in gapped DNA structures.

Here we investigated the ability of the human X-family DNA polymerases beta and lambda to bypass thymine glycol (Tg) in gapped DNA substrates with the damage located in a defined position of the template strand. Maximum velocities and the Michaelis constant values were determined to study DNA synthesis in the presence of either Mg(2+) or Mn(2+). Additionally, the influence of hRPA (human replication protein A) and hPCNA (human proliferating cell nuclear antigen) on TLS (translesion synthesis) activity of DNA polymerases beta and lambda was examined. The results show that (i) DNA polymerase lambda is able to catalyze DNA synthesis across Tg, (ii) the ability of DNA polymerase lambda to elongate from a base paired to a Tg lesion is influenced by the size of the DNA gap, (iii) hPCNA increases the fidelity of Tg bypass and does not influence normal DNA synthesis catalyzed by DNA polymerase lambda, (iv) DNA polymerase beta catalyzes the incorporation of all four dNTPs opposite Tg, and (v) hPCNA as well as hRPA has no specific effect on TLS in comparison with the normal DNA synthesis catalyzed by DNA polymerase beta. These results considerably extend our knowledge concerning the ability of specialized DNA polymerases to cope with a very common DNA lesion such as Tg.

Analysis of specialized DNA polymerases expression in human gliomas: association with prognostic significance.

Aberrant activation of the translesion DNA synthesis (TLS) pathway has been suggested to play a role in tumorigenesis by promoting genetic mutations. We therefore examined glioma specimens for the expression of specialized DNA polymerases involved in TLS and assessed their prognostic significance. The expression levels of DNA polymerase κ (Pol κ), Pol ι, and Pol η were assessed in 40 primary glioma samples and 10 normal brain samples using quantitative real-time PCR and Western blot analysis. Their prognostic significance was evaluated using a population-based tissue microarray derived from a cohort of 104 glioma patients. Overexpression of Pol κ and Pol ι was observed in 57.5% (23-40) and 27.5% (11-40) of patients, respectively, whereas no significant expression of Pol η was seen in the specimens. Immunohistochemical studies revealed positive Pol κ and Pol ι staining in 72 (69.2%) and 33 (31.7%) of the 104 glioma specimens, respectively. Pol κ expression was associated with advanced stages of the disease. Both Pol κ- and Pol ι-positive staining were associated with shorter survival in glioma patients (P < .001 and P = .014, respectively). A multivariate survival analysis identified Pol κ as an independent prognostic factor for glioma patients (P < .001). These findings demonstrate, for the first time, that the expression of Pol κ and Pol ι is deregulated in gliomas, and upregulation of Pol κ is associated with poorer prognosis in glioma patients.

[Research progress on DNA polymerase beta and genetic instability].

DNA polymerase beta (pol beta), a predominant enzyme involved in DNA base excision repair (BER), plays an important role in repair of DNA damage and maintaining stability and integrality of genome. Pol beta lacks 3' to 5' proofreading activity, which contributes to its low fidelity in DNA synthesis. There are different results about action rule of pol beta in genetic instability and mechanism of pol beta in tumorigenesis. The structure and function of pol beta, the relationship between pol beta and genome instability, and abnormal expression and mutation of pol beta in tumor were reviewed.

Human methyl purine DNA glycosylase and DNA polymerase beta expression collectively predict sensitivity to temozolomide.

Overexpression of N-methylpurine DNA glycosylase (MPG) has been suggested as a possible gene therapy approach to sensitize tumor cells to the cell-killing effects of temozolomide, an imidazotetrazine-class chemotherapeutic alkylating agent. In the present study, we show that both elevated MPG expression and short hairpin RNA-mediated loss of DNA polymerase beta (Pol beta) expression in human breast cancer cells increases cellular sensitivity to temozolomide. Resistance to temozolomide is restored by complementation of either wild-type human Pol beta or human Pol beta with an inactivating mutation specific to the polymerase active site yet functional for 5'-deoxyribose-phosphate (5'dRP) lyase activity. These genetic and cellular studies uniquely demonstrate that overexpression of MPG causes an imbalance in base excision repair (BER), leading to an accumulation of cytotoxic 5'dRP lesions, and that the 5'dRP lyase activity of Pol beta is required to restore resistance to temozolomide. These results imply that Pol beta-dependent 5'dRP lyase activity is the rate-limiting step in BER in these cells and suggests that BER is a tightly balanced pathway for the repair of alkylated bases such as N7-methylguanine and N3-methyladenine. Furthermore, we find that 5'dRP-mediated cell death is independent of caspase-3 activation and does not induce the formation of autophagosomes, as measured by green fluorescent protein-light chain 3 localization. The experiments presented herein suggest that it will be important to investigate whether an active BER pathway could be partially responsible for the temozolomide-mediated resistance seen in some tumors and that balanced BER protein expression and overall BER capacity may help predict sensitivity to temozolomide.

Mammary carcinogenesis in transgenic mice expressing a dominant-negative mutant of DNA polymerase beta in their mammary glands.

DNA polymerase beta (polbeta) is a major contributor to mammalian DNA damage repair through its gap-filling DNA synthesis and 5'-deoxyribose phosphate lyase activities. In this way, polbeta plays pivotal roles in the repair of oxidative DNA damage, replication, embryonic survival, neuronal development, meiosis, apoptosis and telomere function. A 36 kDa truncated polbetaDelta protein is expressed in human colorectal, breast, lung and renal carcinomas, but not in normal matched tissues. Interestingly, a binary protein-protein complex of polbetaDelta and X-ray cross-complementing group 1 acts as dominant-negative mutant. In this study, the potential tumorigenic activity of polbetaDelta was examined in nude and transgenic mouse models. Mouse embryonic fibroblasts (MEFs) expressing polbetaDelta in the absence of endogenous polbeta exhibited increased susceptibility to N-methyl-N-nitrosourea (MNU)-induced morphological transformation as compared with cells expressing wild-type (WT) polbeta. This was accompanied by reduced gap-filling DNA synthesis activity. Anchorage-independent transformed cells derived from polbetaDelta-expressing MEFs induced 100% tumor occurrence in nude mice. To support these data, we established transgenic mice expressing polbetaDelta specifically in the mammary glands from a whey acidic protein promoter-driven transgene. This is the first report of transgenic mice with tissue-specific expression of polbetaDelta. MNU-induced tumor formation was analyzed in transgenic mice expressing polbetaDelta together with endogenous WT polbeta in their mammary glands and in normal control mice expressing only WT polbeta. The latent period of tumor appearance was markedly shorter and tumor incidence was significantly higher in transgenic animals than in control animals treated under the same conditions. These results indicate that cells expressing the mutant polbetaDelta display an enhanced sensitivity to MNU that probably underlies an increased susceptibility to tumorigenesis.

Regulation of DNA repair fidelity by molecular checkpoints: "gates" in DNA polymerase beta's substrate selection.

With an increasing number of structural, kinetic, and modeling studies of diverse DNA polymerases in various contexts, a complex dynamical view of how atomic motions might define molecular "gates" or checkpoints that contribute to polymerase specificity and efficiency is emerging. Such atomic-level information can offer insights into rate-limiting conformational and chemical steps to help piece together mechanistic views of polymerases in action. With recent advances, modeling and dynamics simulations, subject to the well-appreciated limitations, can access transition states and transient intermediates along a reaction pathway, both conformational and chemical, and such information can help bridge the gap between experimentally determined equilibrium structures and mechanistic enzymology data. Focusing on DNA polymerase beta (pol beta), we present an emerging view of the geometric, energetic, and dynamic selection criteria governing insertion rate and fidelity mechanisms of DNA polymerases, as gleaned from various computational studies and based on the large body of existing kinetic and structural data. The landscape of nucleotide insertion for pol beta includes conformational changes, prechemistry, and chemistry "avenues", each with a unique deterministic or stochastic pathway that includes checkpoints for selective control of nucleotide insertion efficiency. For both correct and incorrect incoming nucleotides, pol beta's conformational rearrangements before chemistry include a cascade of slow and subtle side chain rearrangements, followed by active site adjustments to overcome higher chemical barriers, which include critical ion-polymerase geometries; this latter notion of a prechemistry avenue fits well with recent structural and NMR data. The chemical step involves an associative mechanism with several possibilities for the initial proton transfer and for the interaction among the active site residues and bridging water molecules. The conformational and chemical events and associated barriers define checkpoints that control enzymatic efficiency and fidelity. Understanding the nature of such active site rearrangements can facilitate interpretation of existing data and stimulate new experiments that aim to probe enzyme features that contribute to fidelity discrimination across various polymerases via such geometric, dynamic, and energetic selection criteria.

Mechanism of adenomatous polyposis coli (APC)-mediated blockage of long-patch base excision repair.

Recently, we found an interaction between adenomatous polyposis coli (APC) and DNA polymerase beta (pol-beta) and showed that APC blocks strand-displacement synthesis of long-patch base excision repair (LP-BER) (Narayan, S., Jaiswal, A. S., and Balusu, R. (2005) J. Biol. Chem. 280, 6942-6949); however, the mechanism is not clear. Using an in vivo LP-BER assay system, we now show that the LP-BER is higher in APC-/- cells than in APC+/+ cells. In addition to pol-beta, the pull-down experiments showed that the full-length APC also interacted with flap endonuclease 1 (Fen-1). To further characterize the interaction of APC with pol-beta and Fen-1, we performed a domain-mapping of APC and found that both pol-beta and Fen-1 interact with a 138-amino acids peptide from the APC at the DRI-domain. Our functional assays showed that APC blocks pol-beta-mediated 1-nucleotide (1-nt) as well as strand-displacement synthesis of reduced abasic, nicked-, or 1-nt gapped-DNA substrates. Further studies demonstrated that APC blocks 5'-flap endonuclease as well as the 5'-3' exonuclease activity of Fen-1 resulting in the blockage of LP-BER. From these results, we concluded that APC can have three different effects on the LP-BER pathway. First, APC can block pol-beta-mediated 1-nt incorporation and strand-displacement synthesis. Second, APC can block LP-BER by blocking the coordinated formation and removal of the strand-displaced flap. Third, APC can block LP-BER by blocking hit-and-run synthesis. These studies will have important implications for APC in DNA damage-induced carcinogenesis and chemoprevention.

DNA polymerase-beta is expressed early in neurons of Alzheimer's disease brain and is loaded into DNA replication forks in neurons challenged with beta-amyloid.

Cultured neurons exposed to synthetic beta-amyloid (Abeta) fragments reenter the cell cycle and initiate a pathway of DNA replication that involves the repair enzyme DNA polymerase-beta (DNA pol-beta) before undergoing apoptotic death. In this study, by performing coimmunoprecipitation experiments on cross-linked nucleoprotein fragments from Abeta-treated neurons, we demonstrate that DNA pol-beta coimmunoprecipitates with cell division cycle 45 (Cdc45) and with DNA primase in short nucleoprotein fragments. This indicates that DNA pol-beta is loaded into neuronal DNA replication forks after Abeta treatment. In response to Abeta the canonical DNA-synthesizing enzyme DNA pol-delta also was loaded into neuronal replication forks, but at later times than DNA pol-beta. Methoxyamine, an inhibitor of the apurinic/apyrimidinic endonuclease that allows for the recruitment of DNA pol-beta during the process of base excision repair (BER), failed to affect coimmunoprecipitation between DNA pol-beta and Cdc45, indicating that DNA pol-beta loading to the replication forks is independent of DNA breaks. However, methoxyamine reduced DNA replication and ensuing apoptosis in neurons exposed to Abeta, suggesting that an efficient BER process allows DNA replication to proceed up to the threshold for death. These data demonstrate that DNA pol-beta is an essential component of the DNA replication machinery in Abeta-treated neurons and additionally support the hypothesis of a close association of cell cycle events with neuronal death in Alzheimer's disease (AD). Accordingly, by investigating the neuronal expression of DNA pol-beta, along with phosphorylated retinoblastoma protein and neurofibrillary changes in AD brain, we show an early involvement of DNA pol-beta in the pathogenesis of AD.

Enhanced expression and activity of DNA polymerase beta in chronic myelogenous leukemia.

BACKGROUND: Chronic myelogenous leukemia (CML) is characterized by an initial chronic phase that invariably evolves to the more aggressive phase of blast crisis. Although the determinants of this transition are still unknown, it has been shown that the blast crisis is accompanied by genetic instability. MATERIALS AND METHODS: The expression and activity of the error-prone DNA polymerase beta (pol beta) were investigated in blood samples from CML patients, by Western blotting and by an in vitro replication assay, respectively. RESULTS: Pol beta expression and activity were significantly higher in CML samples compared to those of healthy donors. CONCLUSION: Our results suggest that the excess of pol beta in CML could contribute to the genetic instability observed during the evolution of the disease from the chronic phase to blast crisis.

[Vector-mediated RNA interference of DNA polymerase beta in human bronchial epithelial cells].

OBJECTIVE: To knock down the expression of polymerase beta gene in human bronchial epithelial cells with technology of vector-mediated RNA interference (RNAi), to provide research tool for the study on the functions and mechanisms of polymerase beta in repairing of DNA damaged by environmental chemical pollutants (ECPs). METHODS: Technology of molecular clone was used to construct the recombination vector of "pEGFP-C1-U6-dsRNA" for the polymerase beta RNAi. The recombinants were transfected into human bronchial epithelial cells with kit of liperfectamine 2000. The control groups included normal human bronchial epithelial cells and human bronchial epithelial cells transfected with "pEGFP-Cl1. Cells were screened by G418, then technology of fluorescence microscopy imaging was used to observe the result of transrfection. The expresison level of polymerase beta was detected by Western blotting. RESULTS: The expression level of polymerase beta in human bronchial epithelial cells transfected with the recombinant of "pEGFP-C1-U6-dsRNA" was about 17.3% of what in the normal cells. CONCLUSION: The RNAi of polymerase beta gene in human bronchial epithelial cells was successful.

Up-regulation of the fidelity of human DNA polymerase lambda by its non-enzymatic proline-rich domain.

DNA repair pathways are essential for maintaining genome stability. DNA polymerase beta plays a critical role in base-excision repair in vivo. DNA polymerase lambda, a recently identified X-family homolog of DNA polymerase beta, is hypothesized to be a second polymerase involved in base-excision repair. The full-length DNA polymerase lambda is comprised of three domains: a C-terminal DNA polymerase beta-like domain, an N-terminal BRCA1 C-terminal domain, and a previously uncharacterized proline-rich domain. Strikingly, pre-steady-state kinetic analyses reveal that, although human DNA polymerase lambda has almost identical fidelity to human DNA polymerase beta, the C-terminal DNA polymerase beta-like domain alone displays a dramatic, up to 100-fold loss in fidelity. We further demonstrate that the non-enzymatic proline-rich domain confers the increase in fidelity of DNA polymerase lambda by significantly lowering incorporation rate constants of incorrect nucleotides. Our studies illustrate a novel mechanism, in which the DNA polymerase fidelity is controlled not by an accessory protein or a proofreading exonuclease domain but by an internal regulatory domain.