Yajing Zhou

Characterization of human DNA polymerase delta and its subassemblies reconstituted by expression in the MultiBac system.

Mammalian DNA polymerase δ (Pol δ), a four-subunit enzyme, plays a crucial and versatile role in DNA replication and DNA repair processes. We have reconstituted human Pol δ complexes in insect cells infected with a single baculovirus into which one or more subunits were assembled. This system allowed for the efficient expression of the tetrameric Pol δ holoenzyme, the p125/p50 core dimer, the core+p68 trimer and the core+p12 trimer, as well as the p125 catalytic subunit. These were isolated in milligram amounts with reproducible purity and specific activities by a highly standardized protocol. We have systematically compared their activities in order to gain insights into the roles of the p12 and p68 subunits, as well as their responses to PCNA. The relative specific activities (apparent k cat) of the Pol δ holoenzyme, core+p68, core+p12 and p125/p50 core were 100, 109, 40, and 29. The corresponding apparent K ds for PCNA were 7.1, 8.7, 9.3 and 73 nM. Our results support the hypothesis that Pol δ interacts with PCNA through multiple interactions, and that there may be a redundancy in binding interactions that may permit Pol δ to adopt flexible configurations with PCNA. The abilities of the Pol δ complexes to fully extend singly primed M13 DNA were examined. All the subassemblies except the core+p68 were defective in their abilities to completely extend the primer, showing that the p68 subunit has an important function in synthesis of long stretches of DNA in this assay. The core+p68 trimer could be reconstituted by addition of p12.

Production of Recombinant Human DNA Polymerase Delta in a Bombyx mori Bioreactor

Eukaryotic DNA polymerase δ (pol δ) plays a crucial role in chromosomal DNA replication and various DNA repair processes. It is thought to consist of p125, p66 (p68), p50 and p12 subunits. However, rigorous isolation of mammalian pol δ from natural sources has usually yielded two-subunit preparations containing only p125 and p50 polypeptides. While recombinant pol δ isolated from infected insect cells have some problems of consistency in the quality of the preparations, and the yields are much lower. To address these deficiencies, we have constructed recombinant BmNPV baculoviruses using MultiBac system. This method makes the generation of recombinant forms of pol δ containing mutations in any one of the subunits or combinations thereof extremely facile. From about 350 infected larvae, we obtained as much as 4 mg of pol δ four-subunit complex. Highly purified enzyme behaved like the one of native form by rigorous characterization and comparison of its activities on poly(dA)/oligo(dT) template-primer and singly primed M13 DNA, and its homogeneity on FPLC gel filtration. In vitro base excision repair (BER) assays showed that pol δ plays a significant role in uracil-intiated BER and is more likely to mediate LP BER, while the trimer lacking p12 is more likely to mediate SN BER. It seems likely that loss of p12 modulates the rate of SN BER and LP BER during the repair process. Thus, this work provides a simple, fast, reliable and economic way for the large-scale production of human DNA polymerase δ with a high activity and purity, setting up a new platform for our further research on the biochemical properties of pol δ, its regulation and the integration of its functions, and how alterations in pol δ function could contribute to the etiology of human cancer or other diseases that can result from loss of genomic stability.

P50, the small subunit of DNA polymerase delta, is required for mediation of the interaction of polymerase delta subassemblies with PCNA.

Mammalian DNA polymerase δ (pol δ), a four-subunit enzyme, plays a crucial and versatile role in DNA replication and various DNA repair processes. Its function as a chromosomal DNA polymerase is dependent on the association with proliferating cell nuclear antigen (PCNA) which functions as a molecular sliding clamp. All four of the pol δ subunits (p125, p50, p68, and p12) have been reported to bind to PCNA. However, the identity of the subunit of pol δ that directly interacts with PCNA and is therefore primarily responsible for the processivity of the enzyme still remains controversial. Previous model for the network of protein-protein interactions of the pol δ-PCNA complex showed that pol δ might be able to interact with a single molecule of PCNA homotrimer through its three subunits, p125, p68, and p12 in which the p50 was not included in. Here, we have confirmed that the small subunit p50 of human pol δ truthfully interacts with PCNA by the use of far-Western analysis, quantitative ELISA assay, and subcellular co-localization. P50 is required for mediation of the interaction between pol δ subassemblies and PCNA homotrimer. Thus, pol δ interacts with PCNA via its four subunits.

Codon optimization enhances protein expression of Bombyx mori nucleopolyhedrovirus DNA polymerase in E. coli.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a major viral agent that causes deadly grasserie disease in silkworms, while BmNPV DNA polymerase (BmNPV-pol), encoded by ORF53 gene, plays a central role in viral DNA replication. Efficacy studies of BmNPV-POL are limited because of poor heterologous protein expression in E. coli. Here, we redesigned the BmNPV-pol to preferentially match codon frequencies of E. coli without altering the amino acid sequence. Following de novo synthesis, codon-optimized BmNPV-pol (co-BmNPV-pol) gene was cloned into pET32a and pGEX-4T-2 vector. The expression of co-BmNPV-POL in E. coli was significantly increased when BmNPV-POL was fused with GST protein rather than a His-tag. The co-BmNPV-POL fusion proteins were isolated using GST affinity chromatography and Mono Q iron exchange chromatography. Protein purity and identity were confirmed by western blot and MALDI-TOF analyses. The biological activity of purified proteins was measured on a poly(dA)/oligo(dT) primer/template. The specific polymerasing activity of the recombinant BmNPV-POL was 6,329 units/mg at optimal conditions. Thus, a large amount of purified protein as a soluble form with high activity would provide many benefits for the functional research and application of BmNPV-POL.

Proteolysis of the Human DNA Polymerase Delta Smallest Subunit p12 by μ-Calpain in Calcium-Triggered Apoptotic HeLa Cells

Degradation of p12 subunit of human DNA polymerase delta (Pol δ) that results in an interconversion between Pol δ4 and Pol δ3 forms plays a significant role in response to replication stress or genotoxic agents triggered DNA damage. Also, the p12 is readily degraded by human calpain in vitro. However, little has been done for the investigation of its degree of participation in any of the more common apoptosis. Here, we first report that the p12 subunit is a substrate of μ-calpain. In calcium-triggered apoptotic HeLa cells, the p12 is degraded at 12 hours post-induction (hpi), restored thereafter by 24 hpi, and then depleted again after 36 hpi in a time-dependent manner while the other three subunits are not affected. It suggests a dual function of Pol δ by its interconversion between Pol δ4 and Pol δ3 that is involved in a novel unknown apoptosis mechanism. The proteolysis of p12 could be efficiently blocked by both calpain inhibitor ALLN and proteasome inhibitor MG132. In vitro pull down and co-immunoprecipitation assays show that the μ-calpain binds to p12 through the interaction of μ-calpain with Pol δ other three subunits, not p12 itself, and PCNA, implying that the proteolysis of p12 by μ-calpain might be through a Pol δ4/PCNA complex. The p12 cleavage sites by μ-calpain are further determined as the location within a 16-amino acids peptide 28-43 by in vitro cleavage assays. Thus, the p12/Pol δ is a target as a nuclear substrate of μ-calpain in a calcium-triggered apoptosis and appears to be a potential marker in the study of the chemotherapy of cancer therapies.

Dry Sliding Wear Characteristics of Ti-6.5Al-3.5Mo-1.5Zr-0.3Si Alloy at Various Sliding Speeds

Dry sliding wear behavior of Ti-6.5Al-3.5Mo-1.5Zr-0.3Si (hereafter called TC11) alloy was investigated under various sliding speeds and distances. TC11 alloy presented marked variations of the wear rate with an increase of sliding speed from 0.5 to 4 m/s. Especially at high loads, a severe-to-mild wear transition occurred with the climax at 2.68 m/s, the lower values were at 0.5 to 1.5 m/s, and the lowest point was at 4 m/s. TC11 alloy also presented a mild-to-severe wear transition at 2.68 m/s with an increase of sliding distance, but not at the others. With a thorough examination for worn surfaces, subsurfaces and wear debris, the distinct characteristics were noticed to correspond to the wear behavior in various conditions. The highest wear rate at 2.68 m/s and the lowest wear rate at 4 m/s, respectively, corresponded to no oxides and large wear debris particles as well as more oxides and small wear debris particles. The tribo-oxides formed at room temperature were suggested to be protective, which seemed to be contradictory to the popular view that tribo-oxides of titanium alloys possess no wear-reducing effect.

Expression, purification, and enzymatic characterization of Bombyx mori nucleopolyhedrovirus DNA polymerase

Bombyx mori nucleopolyhedrovirus (BmNPV) is a major viral agent that causes deadly grasserie disease in silkworms. BmNPV DNA polymerase (Bm-DNAPOL), encoded by the ORF53 gene, plays a central role in viral DNA replication. In this work, a His-tagged Bm-DNAPOL fusion protein, constructed using a novel MultiBac expression system, was overexpressed in Sf-9 insect cells, purified to near homogeneity on Ni-NTA agarose beads and further purified by ion-exchange chromatography. About 0.4 mg of enzyme was obtained from about 1 × 109 infected Sf-9 cells in suspension culture. Characterization of the highly purified enzyme indicated that Bm-DNAPOL is a monomer with an apparent molecular mass of approximately 110,000 Da. It possessed a specific activity of 15,126.3 U/mg under optimal in vitro reaction conditions and behaved in the manner of a proliferating cell nuclear antigen (PCNA)-independent DNA polymerase on both poly(dA)/oligo(dT) primer/template and singly premiered M13 DNA. BmNPV viral replication may be independent of replication factor C and a PCNA complex, while single-stranded DNA binding protein might play an important role in BmNPV DNA replication. These findings will be significant in studies on BmNPV-based disease in silkworms and for using silkworms as a bioreactor for the production of biomolecules of commercial importance.

Nervous necrosis virus capsid protein exploits nucleolar phosphoprotein Nucleophosmin (B23) function for viral replication

Nucleolar proteins facilitate the replication of certain human and animal viruses through interaction with viral proteins. In this study, an interaction between nervous necrosis virus capsid protein and nucleolar phosphoprotein B23 was identified using in vitro experimental approaches. The capsid protein binds to B23 early during the viral infection and accumulates in the nucleus, particularly in the nucleolus. However, over the course of the infection B23 is redistributed from the nucleoli to the nucleoplasm. siRNA-mediated knockdown of B23 reduced viral replication and cytopathic effect. Thus, B23 targets capsid protein to the nucleus and facilitates NNV replication. The results provide the first demonstration that nucleolar protein B23 has a direct role in the nodavirus replication process.

Multiple Forms of Human DNA Polymerase Delta Sub-Assembling in Cellular DNA Transactions.

Among three major replicative DNA polymerases of the B-family, Pol α, Pol δ and Pol ε, Pol δ plays an essential role in chromosomal DNA replication and is also involved in various DNA repair processes in eukaryotes. Human Pol δ is commonly viewed as a heterotetrameric complex, consisting of the catalytic subunit p125 and second subunit p50, together with two additional accessory subunits, p68 and p12. A growing body of research has shown that the latter subunits play a critical role in the regulation of Pol δ functions. The formation of a new form of Pol δ, heterotrimer Pol δ3, is found by virtue of the depletion of p12 through the ubiquitin-proteasome pathway in response to DNA damages that are trigged by UV irradiation, alkylating agents, oxidative and replication stresses. Pol δ3 exhibits significant differences in properties to its progenitor with a major impact on cellular processes in genomic surveillance, DNA replication and DNA repair. Our recent studies indicate that there exists an alternative pathway for Pol δ3 formation by calpain-mediated proteolysis of p12 in a calcium-triggered apoptosis in living cells. In this article, we review and discuss the recent advances from our group and others in the studies of human Pol δ with an emphasis on the generation of its multiple forms by reconstitution and subsequent alternations in enzymatic properties, the multiple pathways of the Pol δ3 formation in living cells, and the phylogenetic analysis of the evolutionary history on POLD4 gene that is for the p12 subunit.

Effect of Various Nanoparticles on Tribo-Layers and Wear Behavior of TC11 Alloy

Multilayer graphene (MLG), Fe2O3, and their nanocomposites with various proportions and amounts were applied as additives and directly participated in the formation of tribo-layers during sliding wear of TC11 alloy against AISI 52100 steel. Their ingredients and amounts were found to exert substantial effects on the additive-containing tribo-layers and wear behavior. Irrespective of the added amount of MLG or Fe2O3, the formed tribo-layers, because of the lack of load-bearing or lubricant capacity, readily lost stability and protection function, causing a high wear rate. However, a small quantity of MLG/Fe2O3 nanocomposites could result in a remarkable decrease in the wear rate. This was attributed to the stable existence and continuous protection of a friction-reduced and wear-resistant double-layer tribo-layer. In particular, Fe2O3-rich nanocomposite additives produced more protective tribo-layers to markedly improve the wear resistance of TC11 alloy.