Nuclear extract, human

Finally, GTP is bound by many proteins, and diazirine-bearing thiol-cleavable GTP analogs (e.g., 23) have been proposed as probes to detect such proteins. By this method, H-Ras was successfully labeled in a nuclear extract of cultured human cells [70]. 

David-Cordonnier M-H, Boiteux S, O Neill P (2001a) Efficiency of excision of 8-oxo-guanine within DNA clustered damage by XRS5 nuclear extracts and purified human OGGI protein. Biochemistry 40 11881-11818... 

It is more likely that p53 influences repair in a regulatory capacity. One link between p53 and NER was made with the observation that the p5 3-regulated Gadd45 binds to PCNA, a component of both replication and repair [206]. Overexpression of Gadd45 provided a small level of protection from cisplatin [207], whereas Gadd45 antisense DNA sensitized human colon carcinoma cells to cisplatin, an effect which was associated with a decrease in repair [201], It was hypothesized that the Gadd45 protein could interact directly with the repair proteins because it stimulated repair synthesis in nuclear extracts [206] however, this result could not be reproduced under a variety of experimental conditions [208][209], Thus, it seems likely that there are still some as yet unidentified factors which link p53 and the excision repair pathway. 

Studies are now underway to elucidate the mechanism of VDR interaction with the VDRE. Liao et al. [251] provided the first evidence that an accessory factor is essential for VDR binding to VDRE, by showing that recombinant VDR does not bind to human osteocalcin VDRE without an accessory factor from nuclear extract. This result was also found by two other independent laboratories [252, 253], The accessory factor, termed nuclear accessory factor (NAF) or receptor auxiliary factor (RAF), is a 55... 

Several topoisomerases have been shown to be substrates for protein kinases. Nuclear extracts from a human cell line contain a protein kinase which phosphorylates DNA topoisomerase I from the same cell line (Mills et al., 1982). The type I topoisomerase purified from Novikoff hepatoma cells was found to be a phosphoprotein (Durban et al., 1983). Treatment with alkaline phosphatase dephosphorylates the enzyme and reduces its DNA-relaxing activity. Subsequent treatment with protein kinase restores the activity of the topoisomerase to its original level (Durban et al., 1983). 

Nealon K, Nicholl ID, Kenny MK (1996) Characterization of the DNA polymerase requirement of human base excision repair. Nucleic Acids Res 24 3763-70 Dianov GL, Price A, Lindahl T (1992) Generation of single nucleotide repair patches following excision of uracil residues from DNA. Mol Cell Biol 12 1605-12 Singhal RK, Prasad R, Wilson SH (1995) DNA polymerase / conducts the gap-filling step in uracU-initiated base excision repair in a bovine testis nuclear extract. J Biol Chem 270 949-57... 

SR-proteins can be expressed as recombinant proteins in Escherichia coli, but they lack the post-translational phosphorylation of the serine residues and are poorly soluble in the absence of chaotropic reagents. It is possible to phosphorylate bacterially produced protein by preincubation in nuclear extract or, even more efficiently, by the addition of purified recombinant SR-protein specific kinases Clk/Sty8 or SRPK.9 Soluble and phosporylated SR-proteins can also be produced in insect cells using the baculovirus system10 although it is not resolved whether these proteins behave exactly as those produced in human cells. 

The ability of human cell (HeLa) extracts to repair the exocydic 4-hydroxy-2-nonenal-dG adducts has been investigated using plasmid DNAs randomly modified by 4-hydroxy-2-nonenal [123]. Incubation of modified plasmids with HeLa nuclear extracts in the presence of [32P]dCTP led to the incorporation of 32P indicative of repair synthesis. 32P-Postlabeling of the modified plasmid revealed that approximately 50-60% of the 4-hydroxy-2-nonenal-dG adducts initially present in the plasmid were removed on incubation with the extracts. This analysis also indicated that two of the four isomeric 4-hydroxy-2-nonenal-dG adducts were preferentially removed. The findings that MjdG, PdG, and the 4-hydroxy-2-none-nal-dG adducts are all repaired by bacterial and mammalian NER suggests that other exocyclic dG adducts are repaired by this pathway. 

Figure 12.5 (a) Autoradiographs of dual excision products NER of (+)-cis-l, (+)-trans-l, and (-)-trans-/ 135mer duplexes in the 5 -.., CCATCG CTACC... sequence context (CC C-I in Figure 12.9) hybridized with a fully complementary strand with C opposite G. The duplexes were internally 32P 5 -end-labeled at the first C shown here at the sixth phosphodiester bond on the 5 -side of the B[o]P-N2-dG lesions (G ). The dual-incision products were obtained after incubation of the 135mer duplexes containing different stereoisomeric G lesions with nuclear extracts from human HeLa cells for 30 min at... 

Fig. 14. Electrophoretic mobility shift assay for the detection of NFkB and AP-1 in human bronchial epithelial cell line BetlA. The human bronchial epithelial cells were preincubated with mac-rolide antibiotics (10 M) 24 hr before stimulation by phorbol myristate acetate (PMA, 10" M). The nuclear extracts were isolated and EMSAs were performed for the detection of (left) NFkB and (right) AP-1. EM and CAM suppressed the activation of both of the transcription factors crucial in the regulation of the IL-8 gene.

Cytoplasmic extract Nuclear extract Normal human sera Calf sera... 

Lee, J. W., Blanco, L., Zhou, T., Garcia-Diaz, M., Bebenek, K., Kunkel, T. A., Wang, Z., and Povirk, L. F. (2004). Implication of DNA polymerase lambda in alignment-based gap filling for nonhomologous DNA end joining in human nuclear extracts. /. Biol. Chem. 279, 805-811. 

Splice with Human Nuclear Extracts in vitro... 

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