Real-Time BIAcore Measurements of Escherichia coli Single-Stranded DNA Binding (SSB) Protein to Polydeoxythymidylic Acid Reveal Single-State Kinetics with Steric Cooperativity

119

The estrogen receptor (ER) belongs to a superfamily of ligand-inducible transcription factors. Functions of these proteins (dimerization, DNA binding, and interaction with other transcription factors) are modulated by binding of their corresponding ligands. It is, however, controversial whether various ER ligands affect the receptor's ability to bind its specific DNA element (ERE).By using real time interaction analysis we have investigated the kinetics of human (h)ER binding to DNA in the absence and presence of 17␤-estradiol, 17␣-ethynyl estradiol, analogs of tamoxifen, raloxifene, and ICI-182,780. We show that ligand binding dramatically influences the kinetics of hER interaction with specific DNA. We have found that binding of estradiol induces the rapid formation of a relatively unstable ER⅐ERE complex, and binding of ICI-182,780 leads to slow formation (k a is approximately 10 times lower) of a stable receptor-DNA complex (k d is almost 2 orders of magnitude lower). Therefore, binding of estradiol accelerates the frequency of receptor-DNA complex formation more than 50-fold, compared with unliganded ER, and more than 1000-fold compared with ER liganded with ICI-182,780. We hypothesize that a correlation exists between the rate of gene transcription and the frequency of receptor-DNA complex formation. We further show that a good correlation exists between the kinetics of hER-ERE interaction induced by a ligand and its biological effect.Steroid hormones are widely distributed small, lipophilic molecules that participate in intracellular communication and control a wide spectrum of developmental and physiological processes. Their effects are mediated by specific intracellular receptors, a family of proteins that are characterized by a high affinity for the corresponding hormones and an ability to discriminate between structurally closely related ligands. These ligand-inducible receptors can modulate transcription of target genes by virtue of their binding to a specific sequence on DNA in target promoters known as hormone response elements. Although distinct proteins, these receptors are members of a large superfamily of steroid hormone receptors and share many common structural and functional features (1-4).Binding of 17␤-estradiol (E 2 ) 1 to estrogen receptor (ER) is followed by a conformational change, leading to dissociation of the receptor from the complex with the heat shock proteins hsp90 and p59 (5, 6), dimerization (7,8), and activation of DNA binding. After DNA binding the activated receptor can interact with basal transcription factors (9). These interactions are thought to stabilize the preinitiation complex at the promoter, allowing RNA polymerase to initiate transcription (10). Recently a number of transcriptional intermediary factors have been identified that can modify estrogen responsiveness, and several of these proteins interact with the ligand binding domain of the ER in a ligand-dependent manner (11-13). It is obvious that the ligand plays a key role in initiating this cascade of events. ...

Section: Discussionmentioning

confidence: 99%

Estrogen Receptor Ligands Modulate Its Interaction with DNA

Cheskis¹,

Karathanasis²,

Lyttle³

1997

119

“…Additionally, the stoichiometry of protein to DNA in the nucleoprotein complex was adjusted by the following relationships: 1 Prot. RU ϭ 0.79 ssDNA RU (22), and 1 Prot. RU ϭ 0.73 dsDNA RU (23).…”

Section: Methodsmentioning

confidence: 99%

Novel DNA Binding Properties of the Mcm10 Protein from Saccharomyces cerevisiae

Eisenberg

Korza

Carson

et al. 2009

The Mcm10 protein is essential for chromosomal DNA replication in eukaryotic cells. We purified the Saccharomyces cerevisiae Mcm10 (ScMcm10) and characterized its DNA binding properties. Electrophoretic mobility shift assays and surface plasmon resonance analysis showed that ScMcm10 binds stably to both double strand (ds) DNA and single strand (ss) DNA. On short DNA templates of 25 or 50 bp, surface plasmon resonance analysis showed a ϳ1:1 stoichiometry of ScMcm10 to dsDNA. On longer dsDNA templates, however, multiple copies of ScMcm10 cooperated in the rapid assembly of a large, stable nucleoprotein complex. The amount of protein bound was directly proportional to the length of the DNA, with an average occupancy spacing of 21-24 bp. This tight spacing is consistent with a nucleoprotein structure in which ScMcm10 is aligned along the helical axis of the dsDNA. In contrast, the stoichiometry of ScMcm10 bound to ssDNA of 20 -50 nucleotides was ϳ3:1 suggesting that interaction with ssDNA induces the assembly of a multisubunit ScMcm10 complex composed of at least three subunits. The tight packing of ScMcm10 on dsDNA and the assembly of a multisubunit complex on ssDNA suggests that, in addition to protein-DNA, protein-protein interactions may be involved in forming the nucleoprotein complex. We propose that these DNA binding properties have an important role in (i) initiation of DNA replication and (ii) formation and maintenance of a stable replication fork during the elongation phase of chromosomal DNA replication.

“…This value was calculated from the mass ratio, which is given by (RU exp )/(0.79 ϫ RU DNA ), where RU exp is the output signal due to protein binding and RU DNA corresponds to the amount of DNA bound to the chip in resonance units. The factor 0.79 corrects for the fact that the refractive index increment for a typical protein is 79% of that obtained with an equivalent mass of DNA (25). Mass ratio values were converted to molar ratios using the molecular weights of the DNA and protein in question.…”

Section: P]homoduplex or [mentioning

confidence: 99%

DNA Chain Length Dependence of Formation and Dynamics of hMutSα·hMutLα·Heteroduplex Complexes

Blackwell¹,

Wang²,

Modrich³

2001

Formation of a ternary complex between human MutS␣, MutL␣, and heteroduplex DNA has been demonstrated by surface plasmon resonance spectroscopy and electrophoretic gel shift methods. Formation of the hMutL␣⅐hMutS␣⅐heteroduplex complex requires a mismatch and ATP hydrolysis, and depends on DNA chain length. Ternary complex formation was supported by a 200-base pair G-T heteroduplex, a 100-base pair substrate was somewhat less effective, and a 41-base pair heteroduplex was inactive. As judged by surface plasmon resonance spectroscopy, ternary complexes produced with the 200-base pair G-T DNA contained ϳ0.8 mol of hMutL␣/mol of heteroduplex-bound hMutS␣. Although the steady-state levels of the hMutL␣⅐hMutS␣⅐ heteroduplex were substantial, this complex was found to turn over, as judged by surface plasmon resonance spectroscopy and electrophoretic gel shift analysis. With the former method, the majority of the complexes dissociated rapidly upon termination of protein flow, and dissociation occurred in the latter case upon challenge with competitor DNA. However, ternary complex dissociation as monitored by gel shift assay was prevented if both ends of the heteroduplex were physically blocked with streptavidin⅐biotin complexes. This observation suggests that, like hMutS␣, the hMutL␣⅐hMutS␣ complex can migrate along the helix contour to dissociate at DNA ends.The MutS and MutL homologs, which are required for the initiation of mismatch repair, have been implicated in the correction of DNA biosynthetic errors, the transcription-coupled repair of DNA damage, and the fidelity of genetic recombination (1-6). In mammalian cells, MutS␣ (MSH2⅐MSH6 heterodimer), MutS␤ (MSH2⅐MSH3 heterodimer), and MutL␣ (MLH1⅐PMS2 heterodimer) are also thought to function as lesion sensors for certain types of DNA damage that kill by activating apoptosis (2, 6, 7).Repair in the Escherichia coli system is initiated by the binding of MutS to a mismatch (8 -10). Formation of a MutL⅐MutS⅐heteroduplex complex has been demonstrated by DNase I footprint analysis (11), electron microscopy (12), and surface plasmon resonance spectroscopy (SPRS) 1 (13), with assembly of this ternary complex being ATP-dependent. Several lines of evidence indicate that assembly of the ternary complex is required for subsequent steps in mismatch repair. Both MutS and MutL are required for the mismatch-dependent activation of the d(GATC) endonuclease activity of MutH, which cleaves the unmethylated strand of a hemimethylated d(GATC) site, with the ensuing strand break serving to direct repair to the unmethylated DNA strand (14). MutS and MutL are also required for the mismatch-dependent activation of DNA helicase II, which enters the helix at the strand break and initiates the excision step of repair (15). Formation of a MutL␣⅐MutS␣⅐heteroduplex complex has been demonstrated by electrophoretic gel shift analysis with yeast mismatch repair proteins using synthetic heteroduplexes of ϳ50 base pairs (bp) in size (16, 17). However, using gel shift methods and surface plasmon reson...