We have investigated whether DNA polymerase IV (Pol IV; the dinB gene product) contributes to the error rate of chromosomal DNA replication in Escherichia coli. We compared mutation frequencies in mismatch repair-defective strains that were either dinB positive or dinB deficient, using a series of mutational markers, including lac targets in both orientations on the chromosome. Virtually no contribution of Pol IV to the chromosomal mutation rate was observed. On the other hand, a significant effect of dinB was observed for reversion of a lac allele when the lac gene resided on an F(pro-lac) episome.Several mechanisms control the fidelity of the DNA replication process. These include correct base selection by the DNA polymerase, removal of base insertion errors by 3Ј-exonucleolytic proofreading, and correction by DNA mismatch repair (29). In Escherichia coli, base selection and proofreading are performed by the DNA polymerase III (Pol III) holoenzyme, the enzyme that replicates the bacterial chromosome. It is generally considered a highly accurate enzyme (29). Mismatch repair is performed by the mutHLS mismatch repair system (17). In combination, these three processes yield an error rate of 10 Ϫ9 to 10 Ϫ11 error per base pair replicated per cell division (6,29).In addition to Pol III, E. coli possesses four other DNA polymerases, Pol I, Pol II, Pol IV, and Pol V, whose precise functions are still being defined. Pol IV and Pol V belong to the recently described Y family of DNA polymerases

Highly localized radiotherapy with radionuclides is a commonly used treatment modality for patients with unresectable solid tumors. Herein, we propose a novel α-nanobrachytherapy approach for selective therapy of human epidermal growth factor receptor 2 (HER2)-positive breast cancer. This uses local intratumoral injection of 5-nm-diameter gold nanoparticles (AuNPs) labeled with an α-emitter (211At), modified with polyethylene glycol (PEG) chains and attached to HER2-specific monoclonal antibody (trastuzumab). The size, shape, morphology, and zeta potential of the 5 nm synthesized AuNPs were characterized by TEM (Transmission Electron Microscopy) and DLS (Dynamic Light Scattering) techniques. The gold nanoparticle surface was modified by PEG and subsequently used for antibody immobilization. Utilizing the high affinity of gold for heavy halogens, the bioconjugate was labelled with 211At obtained by α irradiation of the bismuth target. The labeling yield of 211At was greater than 99%. 211At bioconjugates were stable in human serum. Additionally, in vitro biological studies indicated that 211At-AuNP-PEG-trastuzumab exhibited higher affinity and cytotoxicity towards the HER2-overexpressing human ovarian SKOV-3 cell line than unmodified nanoparticles. Confocal and dark field microscopy studies revealed that 211At-AuNP-PEG-trastuzumab was effectively internalized and deposited near the nucleus. These findings show promising potential for the 211At-AuNP-PEG-trastuzumab radiobioconjugate as a perspective therapeutic agent in the treatment of unresectable solid cancers expressing HER2 receptors.

The dnaX36(TS) mutant of Escherichia coli confers a distinct mutator phenotype characterized by enhancement of transversion base substitutions and certain (؊1) frameshift mutations. Here, we have further investigated the possible mechanism(s) underlying this mutator effect, focusing in particular on the role of the various E. coli DNA polymerases. The dnaX gene encodes the subunit of DNA polymerase III (Pol III) holoenzyme, the enzyme responsible for replication of the bacterial chromosome. The dnaX36 defect resides in the C-terminal domain V of , essential for interaction of with the ␣ (polymerase) subunit, suggesting that the mutator phenotype is caused by an impaired or altered ␣-interaction. We previously proposed that the mutator activity results from aberrant processing of terminal mismatches created by Pol III insertion errors. The present results, including lack of interaction of dnaX36 with mutM, mutY, and recA defects, support our assumption that dnaX36-mediated mutations originate as errors of replication rather than DNA damagerelated events. Second, an important role is described for DNA Pol II and Pol IV in preventing and producing, respectively, the mutations. In the system used, a high fraction of the mutations is dependent on the action of Pol IV in a (dinB) gene dosage-dependent manner. However, an even larger but opposing role is deduced for Pol II, revealing Pol II to be a major editor of Pol III mediated replication errors. Overall, the results provide insight into the interplay of the various DNA polymerases, and of subunit, in securing a high fidelity of replication.The mechanisms by which cells produce mutations, or try to avoid making them, are of significant research interest. Mutations may occur from replication errors, as DNA replication proceeds with high but not infinite accuracy. While the fidelity of individual DNA polymerases, including their base insertion fidelity and proofreading ability, has been investigated in detail (for reviews, see references 43 and 44), recent emphasis has shifted to the fidelity of the chromosomal replisomes, the multisubunit complexes that perform the simultaneous replication of leading and lagging strands. Specific issues of interest are the contribution of the various replisomal subunits, the mechanisms underlying the differential fidelity of leading and lagging strand replication, and the involvement of the additional DNA polymerases that have been discovered in recent years.In the model system Escherichia coli, chromosomal replication is performed by the 17-subunit protein complex DNA polymerase III (Pol III) holoenzyme (HE) (49,50,56). HE is organized into several functional modules: two Pol III core units (one for each strand), two ␤-clamp processivity factors, and the DnaX complex. Each Pol III core is made up of three subunits (␣, ε, and ), in which ␣ is the DNA polymerase, ε is the proofreading subunit (3Ј35Ј exonuclease), and is a stabilizing factor for the ε subunit (37, 82). Each ␤-clamp is a dimer of identical subunits (␤ 2 ) in the shape ...

Podoplanin (PDPN), a mucin-type transmembrane glycoprotein specific to the lymphatic system is expressed in a variety of human cancers, and is regarded as a factor promoting tumor progression. The purpose of this study was to elucidate the molecular role of PDPN in the biology of thyroid cancer cells. PDPN expression was evaluated in primary thyroid carcinomas and thyroid carcinoma cell lines by RT-qPCR, Western blotting, IF and IHC. To examine the role of podoplanin in determining a cell's malignant potential (cellular migration, invasion, proliferation, adhesion, motility, apoptosis), a thyroid cancer cell line with silenced PDPN expression was used. We observed that PDPN was solely expressed in the cancer cells of 40% of papillary thyroid carcinoma (PTC) tissues. Moreover, PDPN mRNA and protein were highly expressed in PTC-derived TPC1 and BcPAP cell lines but were not detected in follicular thyroid cancer derived cell lines. PDPN knock-down significantly decreased cellular invasion, and modestly reduced cell migration, while proliferation and adhesion were not affected. Our results demonstrate that PDPN mediates the invasive properties of cells derived from papillary thyroid carcinomas, suggesting that podoplanin might promote PTC progression.