Ethidium bromide provides a simple tool for identifying genuine DNA-independent protein associations.

Abstract

DNA-dependent and DNA-independent associations of DNA-binding proteins are important in transcriptional regulation. The analysis of DNA-independent associations frequently relies on assaying protein interaction in the absence of target DNA sequences. We have found that contaminating DNA in protein preparations can stabilize DNAdependent associations that may appear DNA-independent. Three cellular proteins of 70, 85, and 110 kDa coimmunoprecipitated with the octamer motif-binding protein Oct-2 because of the presence of contaminating DNA in the cell extracts. In addition, heterodimer formation between Oct-i (or Oct-2) and Pit-i during protein-affinity chromatography was stabilized by the contaminating DNA. In both instances, these DNAdependent protein associations were selectively inhibited by ethidlum bromide in the precipitation reaction without any evident effect on DNA-independent protein associations. Thus, ethidlum bromide may serve as a simple and general indicator of DNA-dependent and DNA-independent protein associations.Transcriptional regulation depends not only on the interactions between sequence-specific DNA-binding proteins and their respective cis-regulatory elements but also on the interactions among these proteins and with other components of the transcriptional machinery (reviewed in refs. 1-3). The availability of tools for studying sequence-specific transcription factors (e.g., cDNA clones and antibodies) has allowed more detailed analysis of the mechanisms by which proteinprotein interactions, both DNA-dependent and DNAindependent, regulate transcription. Current methods for determining DNA-independent protein-protein interactions include coimmunoprecipitation and protein-affinity chromatography. We have used coimmunoprecipitation to identify proteins that associate with the octamer motif-binding proteins Oct-1 (OTF-1, NFIII) and Oct-2 (OTF-2). These transcription factors are particularly interesting because they display the same DNA-binding specificity (4) and share very similar DNA-binding POU domains (5, 6) but display qualitatively different RNA polymerase II transcriptional activation properties. Oct-1 is an effective activator of small nuclear RNA-type promoters, whereas Oct-2 is an effective activator of mRNA-type promoters (7). Therefore, it is likely that Oct-1 and Oct-2 interact preferentially with different components of the transcriptional machinery.We identified four cellular proteins of 68, 70, 85, and 110 kDa that coimmunoprecipitated with Oct-2 from labeled cell extracts in what appeared to be a DNA-independent manner. We noticed, however, that the 70-, 85-, and 110-kDa proteins could bind DNA on their own. We subsequently identified the 70-and 85-kDa proteins as the two heterologous subunits of the human autoantigen Ku, which possesses strong nonspecific DNA-binding properties (ref. 8 and references therein). This observation suggested that the association of these three proteins with Oct-2 might be mediated by contaminating DNA in the labeled cell extracts. Consisten...

Cell polarity protein Par3 complexes with DNA-PK via Ku70 and regulates DNA double-strand break repair

“…Reciprocally, when endogenous Par3 was precipitated, Ku proteins were also specifically co-precipitated. Since Ku proteins are DNA-binding proteins, to exclude the possibility that DNA mediates the interaction between Par3 and Ku70/Ku80, we treated the Par3 immunoprecipitates with micrococcal nuclease, or added ethidium bromide (EB), which is known to disrupt protein-DNA interactions [20], to the lysates before performing immunoprecipitation. Our data showed that DNA did not mediate Par3-Ku association (Supplementary information, Figure S2).…”

An interaction between the DNA-binding domains of RelA(p65) and Sp1 mediates human immunodeficiency virus gene activation.

“…5A). In addition, this specific interaction between RelA and Spl was observed in the presence of ethidium bromide (data not shown), which has been shown to disrupt DNA-dependent protein-protein interactions (37 to the zinc finger (F) region of Spl alone and showed nonspecific binding to the D domain (Fig. 5C).…”

Activation of PARP-1 in response to bleomycin depends on the Ku antigen and protein phosphatase 5

“…Extensive PARP-1 automodification did not seem to be required for Ku binding, as addition of the PARP-1 enzymatic inhibitor 4ANI, which effectively blocked the shift in PARP-1 mobility on SDS-PAGE ( Figure 5c) and inhibited 32 P NAD incorporation by over 95% (data not shown), enhanced the coimmunoprecipitation of PARP-1 with Ku, suggesting a preferential interaction between Ku and PARP-1 before its auto-ADP ribosylation, but subsequent to DNA damage. Finally, addition of ethidium bromide to the extracts, which disrupts protein-DNA interactions (Lai and Herr, 1992), prevented coimmunoprecipitation of PARP-1 and Ku (Figure 5d). Although this result indicates that DNA contact by PARP-1 and/or Ku is important to detect their physical association, it leaves open whether the two factors are preferentially recruited to the same DNA ends or whether the DNA binding of one or both factors is required for their physical interaction.…”