Electrophoretic Mobility Shift Assays for the Analysis of DNA-Protein Interactions

PURPOSE. Poly(ADP-ribosyl)ation is a reversible post-translational modification that requires the contribution of the enzymes poly(ADP-ribose) polymerase-1 (PARP-1) and poly(ADP-ribose) glycohydrolase (PARG). Our study explores expression and activity of PARP-1 and PARG in uveal melanoma cell lines with varying tumorigenic properties.METHODS. Gene profiling on microarrays was conducted using RNA prepared from the uveal melanoma cell lines T97, T98, T108, and T115. The activity of PARP-1 and PARG was monitored by enzymatic assays, whereas their expression was measured by Western blot and PCR. The PARG promoter was analyzed using promoter deletions and site-specific mutagenesis in transfection analyses. The transcription factors binding the PARG promoter were studied by electrophoretic mobility shift assay (EMSA) analyses. Suppression of PARP-1 and PARG expression was performed in T97 and T115 cells by RNAi, and their tumorigenic properties monitored by injections into athymic mice.RESULTS. Expression of PARP-1 was found to vary considerably between uveal melanoma cell lines with distinctive tumorigenic properties in vivo. Sp1 and the ETS protein ERM were shown to bind to the PARG gene promoter to ensure basal transcription in uveal melanoma. Importantly, suppression of PARG gene expression in T97 and T115 cells increased their capacity to form tumors in athymic mice, whereas suppression of PARP-1 significantly reduced or almost entirely abolished tumor formation. CONCLUSIONS.Our results suggest that while overexpression of PARP-1 may confer a proliferative advantage to aggressive uveal melanoma tumors, PARG may, on the other hand, support a tumor suppressor function in vivo. (Invest Ophthalmol Vis Sci.

show abstract

“…30 Gels were dried and autoradiographed at À808C overnight to reveal the position of the shifted DNA-protein complexes generated.…”

Section: Nuclear Extracts and Electrophoretic Mobility Shift Assay (Ementioning

confidence: 99%

Altered Expression of the Poly(ADP-Ribosyl)ation Enzymes in Uveal Melanoma and Regulation ofPARGGene Expression by the Transcription Factor ERM

Molloy-Simard

St-Laurent

Vigneault

et al. 2012

Invest. Ophthalmol. Vis. Sci.

Self Cite

View full text Add to dashboard Cite

show abstract

“…Mycobacterium tuberculosis AcrR contains nine a-helices and two 3 10 -helices (g) in the following order: a1 (residues: [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28], a2 (residues: 37-44), a3 (residues: [48][49][50][51][52][53][54][55], a4 (residues: 58-75), g1 (residues: 80-82), a5 (residues: 91-103), a6 (residues: 107-116), a7 (residues: 123-144), g2 (residues: 145-147), a8 (residues: 154-181), and a9 (residues: 188-207; Fig. 1B).…”

Section: Resultsmentioning

confidence: 99%

The crystal structure of AcrR from Mycobacterium tuberculosis reveals a one‐component transcriptional regulation mechanism

et al. 2019

View full text Add to dashboard Cite

Transcriptional regulator proteins are closely involved in essential survival strategies in bacteria. AcrR is a one‐component allosteric repressor of the genes associated with lipid transport and antibiotic resistance. When fatty acid ligands bind to the C‐terminal ligand‐binding cavity of AcrR, a conformational change in the N‐terminal operator‐binding region of AcrR is triggered, which releases the repressed DNA and initiates transcription. This paper focuses on the structural transition mechanism of AcrR of Mycobacterium tuberculosis upon DNA and ligand binding. AcrR loses its structural integrity upon ligand‐mediated structural alteration and bends toward the promoter DNA in a more compact form, initiating a rotational motion. Our functional characterization of AcrR and description of the ligand‐ and DNA‐recognition mechanism may facilitate the discovery of new therapies for tuberculosis.

show abstract

“…EMSAs were conducted as described in previous studies (Gaudreault et al ., ; Lin et al ., ) with some modifications. DNA fragments for EMSA were obtained by annealing commercially synthesized complementary oligonucleotides (by heating to 65°C and then cooling to room temperature) or by PCR (with primers listed in http://onlinelibrary.wiley.com/doi/10.1111/mmi.12614/suppinfo).…”

Section: Methodsmentioning

confidence: 99%

CRP represses the CRISPR/Cas system in Escherichia coli: evidence that endogenous CRISPR spacers impede phage P1 replication

Yang

Chen

Huang

et al. 2014

Molecular Microbiology

View full text Add to dashboard Cite

SummaryThe CRISPR/Cas system is an important aspect in bacterial immunology. The anti-phage activity of the CRISPR system has been established using synthetic CRISPR spacers, but in vivo studies of endogenous CRISPR spacers are relatively scarce. Here, we showed that bacteriophage P1 titre in Escherichia coli decreased in the glucose-containing medium compared with that in the absence of glucose. This glucose effect of E. coli against phage P1 infection disappeared in cse3 deletion mutants. The effect on the susceptibility to phage P1 was associated with cAMP receptor protein (CRP)-mediated repression of cas genes transcription and crRNA maturation. Analysis of the regulatory element in the cse1 promoter region revealed a novel CRP binding site, which overlapped with a LeuO binding site. Furthermore, the limited sequence identity between endogenous spacers and the phage P1 genome was necessary and sufficient for CRISPRmediated repression of phage P1 replication. Transexpression of the third and seventh spacers in the CRISPR I region or third and sixth spacers in the CRISPR II region effectively reduced phage P1 titres in the CRISPR deletion mutants. These results demonstrate a novel regulatory mechanism for cas repression by CRP and provide evidence that endogenous spacers can repress phage P1 replication in E. coli.

show abstract

Electrophoretic Mobility Shift Assays for the Analysis of DNA-Protein Interactions

Cited by 38 publications

References 56 publications

Altered Expression of the Poly(ADP-Ribosyl)ation Enzymes in Uveal Melanoma and Regulation ofPARGGene Expression by the Transcription Factor ERM

Altered Expression of the Poly(ADP-Ribosyl)ation Enzymes in Uveal Melanoma and Regulation ofPARGGene Expression by the Transcription Factor ERM

The crystal structure of AcrR from Mycobacterium tuberculosis reveals a one‐component transcriptional regulation mechanism

CRP represses the CRISPR/Cas system in Escherichia coli: evidence that endogenous CRISPR spacers impede phage P1 replication

Contact Info

Product

Resources

About