Elucidating the mechanism of DNA-dependent ATP hydrolysis mediated by DNA-dependent ATPase A, a member of the SWI2/SNF2 protein family

The active DNA-dependent ATPase A domain (ADAAD), a member of the SWI2/SNF2 family, has been shown to bind DNA in a structure-specific manner, recognizing DNA molecules possessing double-stranded to single-stranded transition regions leading to ATP hydrolysis. Extending these studies we have delineated the structural requirements of the DNA effector for ADAAD and have shown that the single-stranded and double-stranded regions both contribute to binding affinity while the double-stranded region additionally plays a role in determining the rate of ATP hydrolysis. We have also investigated the mechanism of interaction of DNA and ATP with ADAAD and shown that each can interact independently with ADAAD in the absence of the other. Furthermore, the protein can bind to dsDNA as well as ssDNA molecules. However, the conformation change induced by the ssDNA is different from the conformational change induced by stem-loop DNA (slDNA), thereby providing an explanation for the observed ATP hydrolysis only in the presence of the double-stranded:single-stranded transition (i.e. slDNA)

Calculation of K_SV1and K_SV2 for Protein-ATP-DNA-inhibitor interaction.

*<p>Data reported in Nongkhlaw <i>et al</i>. (Nongkhlaw, 2009).</p>1<p>Average of at least two independent experiments with each experiment done in duplicates.</p

ADAADi binds to Motif Ia of ADAAD leading to conformational changes in the protein.

<p>(A). MAD28 (0.5 μM and 1.0 μM) was titrated with increasing concentration of ADAADiN. (B). MAD8 (0.5 μM) was titrated with increasing concentration of ADAADiN. (C). <b>Electrophoretic mobility shift assay</b>. Lane 1: Free slDNA; Lane 2: slDNA with 1 μM ADAADiN; Lane 3: slDNA with ADAAD; Lane 4: slDNA with ADAAD and 15 μM unlabeled slDNA; Lane 5: slDNA with ADAAD and 1 μM ADAADiN; Lane 6: slDNA with ADAAD and 2 μM ADAADiN. (D). Lane 1: Free slDNA; Lane 2: slDNA with 1 μM ADAADiN; Lane 3: slDNA with ADAAD; Lane 4: slDNA with ADAAD and 4 μM ADAADiN; Lane 5: slDNA with ADAAD and 8 μM ADAADiN; Lane 6: slDNA with ADAAD and 10 μM ADAADiN. (E). <b>Stern-Volmer plots</b>. ADAAD (0.5 μM) was titrated with increasing concentration of acrylamide in presence of either 2 μM ADAADiN (▪), or 2 μM ADAADiN, 3 μM slDNA and 40 μM ATP (•), or 2 μM ADAADiN, 40 μM ATP and 3 μM slDNA (▴), or 40 μM ATP, 2 μM ADAADiN and 3 μM slDNA (▾), or 3 μM slDNA, 2 μM ADAADiN, and 40 μM ATP (⧫). (F). The CD spectra of ADAAD in the absence and presence of ADAADiN (0.2 μM), ATP (20 μM), and slDNA (2 μM) recorded at 25°C.</p

Global Epigenetic Changes Induced by SWI2/SNF2 Inhibitors Characterize Neomycin-Resistant Mammalian Cells

<div><h3>Background</h3><p>Previously, we showed that aminoglycoside phosphotransferases catalyze the formation of a specific inhibitor of the SWI2/SNF2 proteins. Aminoglycoside phosphotransferases, for example neomycin-resistant genes, are used extensively as selection markers in mammalian transfections as well as in transgenic studies. However, introduction of the neomycin-resistant gene is fraught with variability in gene expression. We hypothesized that the introduction of neomycin-resistant genes into mammalian cells results in inactivation of SWI2/SNF2 proteins thereby leading to global epigenetic changes.</p> <h3>Methodology</h3><p>Using fluorescence spectroscopy we have shown that the inhibitor, known as <u>A</u>ctive <u>D</u>NA-<u>d</u>ependent <u>A</u>TPase <u>A</u><u>D</u>omain inhibitor (ADAADi), binds to the SWI2/SNF2 proteins in the absence as well as presence of ATP and DNA. This binding occurs via a specific region known as Motif Ia leading to a conformational change in the SWI2/SNF2 proteins that precludes ATP hydrolysis. ADAADi is produced from a plethora of aminoglycosides including G418 and Streptomycin, two commonly used antibiotics in mammalian cell cultures. Mammalian cells are sensitive to ADAADi; however, cells stably transfected with neomycin-resistant genes are refractory to ADAADi. In resistant cells, endogenous SWI2/SNF2 proteins are inactivated which results in altered histone modifications. Microarray data shows that the changes in the epigenome are reflected in altered gene expression. The microarray data was validated using real-time PCR. Finally, we show that the epigenetic changes are quantized.</p> <h3>Significance</h3><p>The use of neomycin-resistant genes revolutionized mammalian transfections even though questions linger about efficacy. In this study, we have demonstrated that selection of neomycin-resistant cells results in survival of only those cells that have undergone epigenetic changes, and therefore, data obtained using these resistant genes as selection markers need to be cautiously evaluated.</p> </div

Expression of endogenous SG2NA is influenced by ADAADi production.

<p>The transcript as well as protein expression was monitored in the untransfected cells as well as in cells stably transfected with pcDNA 3.1 myc/his (−) vector at passage 13. (A). Endogenous SG2NA transcript was analyzed by quantitative RT-PCR in untransfected and transfected Neuro 2Acells. (B). Endogenous SG2NA protein analyzed by western blot using antibody against SG2NA. (C). <i>sg2na</i> promoter occupancy by RNAPII, Brg1, H3K9Ac, and H3K9Me2 was analysed in untransfected and transfected Neuro 2A cells using ChIP. Fold enrichment was calculated with respect to the mock ChIP done using IgG antibodies. (D). SG2NA transcript level in transfected cells at passage 4. (E). <b>Expression of exogenous SG2NA expressed using pcDNA 3.1 myc/his (−) vector </b><b>is also influenced by ADAADi production.</b> Overexpression of three variants of SG2NA in Neuro2A cells were monitored using anti-myc antibody. Transfected cells (passage 4) were grown as indicated for 12 hours before analysis. Two clones of 87 kDa (87.1 and 87.2), one clone each of 78 kDa (78.1) and of 52 kDa (52.2) were analyzed for protein expression. The cells transfected with vector alone were used as control. Protein expression was observed only when cells were grown in the absence of both antibiotics. (F). Western blot analysis of expression of 87- and 78-kDa proteins in clones 87.1 and 78.1 in stably transfected cells grown in the presence of antibiotics using anti-myc antibody. Lane 1: vector alone transfected cells; Lane 2: 78.1 clone; Lane 3: 87.1 clone. N.S., indicating non-specific band, was used as loading control. (G). The expression of 78- and 87- kDa protein in 78.1, 78.2, 87.1 and 87.2 clones was monitored in stably transfected cells grown in the absence of antibiotics. Lane 1: vector transfected cells; Lane 2: 78.1 clone; Lane 3: 78.2 clone; M: marker; Lane 4: 87.1 clone; Lane 5: 87.2 clone. N.S., indicating non-specific band, was used as loading control. (H). Semi-quantitative RT-PCR analysis done using insert-specific forward primer and vector-specific reverse primer confirms that 87 kDa transcript expression is observed only when the cells are grown the absence of antibiotics. (I). The expression of 87 kDa, 78 kDa, and 52 kDa was not observed in stably transfected cells even after removal of antibiotics when the cells were freeze-thawed at passage 9. (J). Semi-quantitative RT-PCR analysis of APH transcript. Lane1: Untransfected Neuro2A cells; Lane 2: Stably transfected Neuro2A cells; Lane 3: control reaction using purified pcDNA 3.1 myc/his (−) vector.</p

ADAADi formation is catalyzed by different isoforms of APH using different aminoglycoside substrates. (A). APH (3′)-I, APH (3′)-IIa, and APH (3′)-IIIa catalyze ADAADi formation.

<p>ADAADi, synthesized by the three isozymes of APH, was purified and ATPase assays with 0.22 μM His-ADAAD and 68 μM kanamycin (Kan), 44 μM neomycin (Neo), 1.6 μM ADAADiK from APH(3′)-I (I) and APH(3′)-IIa (IIa), 1.2 μM ADAADiK from APH(3′)-IIIa (IIIa), 1.6 μM ADAADiN from APH (3′)-I (I), 2 μM ADAADiN from APH(3′)-IIa (IIa) , and 1.4 μM ADAADiN from APH(3′)-IIIa (IIIa) were done as described. (B). <b>ADAADi is produced from G418 as well as streptomycin</b> by APH (3′)-IIIa. ATPase assays were done either in the absence or presence of 200 μM streptomycin, 2 μM G418, 4 μM ADAADiS, 4 μM ADAADiG418. (C). ADAADi produced using APH (3′)-IIIa from commercially available aminoglycosides.</p

Effect of ADAADi on untransfected and transfected mammalian cells.

<p>(A). Untransfected Neuro2A cells treated with neomycin (•) or ADAADiN (○). (B). Untransfected Neuro2A cells treated with G418 (•) or ADAADiG418 (○). (C). Untransfected Neuro2A cells treated with kanamycin (•) or ADAADiK (○). (D). Comparing the effect of 50 μM ADAADiN on untransfected and stably transfected Neuro2A cells grown as indicated post-selection. (E). Parental C2C12 cells grown in the absence (•) and presence (○) of 200 μM ADAADiK. (F). Puromycin-resistant C2C12 cells carrying the pCPLX retroviral vector and grown in the absence (▾) and presence (▿) of 200 μM ADAADiK. (G). G418-resistant C2C12 cells carrying the pLNCX retroviral vector and grown in the absence (▪) and presence (□) of 200 μM ADAADiK.</p