The Role of the Minor Groove Substituents in Indirect Readout of DNA Sequence by 434 Repressor

Abstract

The sequence of non-contacted bases at the center of the 434 repressor binding site affects the strength of the repressor-DNA complex by influencing the structure and flexibility of DNA (Koudelka, G. B., and Carlson, P. (1992) Nature 355, 89 -91). We synthesized 434 repressor binding sites that differ in their central sequence base composition to test the importance of minor groove substituents and/or the number of base pair hydrogen bonds between these base pairs on DNA structure and strength of the repressor-DNA complex. We show here that the number of base pair H-bonds between the central bases apparently has no role in determining the relative affinity of a DNA site for repressor. Instead we find that the affinity of DNA for repressor depends on the absence or presence the N2-NH 2 group on the purine bases at the binding site center. The N2-NH 2 group on bases at the center of the 434 binding site appears to destabilize 434 repressor-DNA complexes by decreasing the intimacy of the specific repressor-DNA contacts, while increasing the reliance on protein contacts to the DNA phosphate backbone. Thus, the presence of an N2-NH 2 group on the purines at the center of a binding site globally alters the precise conformation of the protein-DNA interface.It is well established that the sequence-specific binding of proteins to DNA involves specific contacts between DNA bases and protein side chains in a process known as "direct-readout." The precise alignment of DNA and protein can be specifically modulated by the sequence of DNA bases in the binding site that are not directly contacted by the protein. This phenomenon is known as indirect readout. In indirect readout, the affinity or specificity of a protein-DNA complex depends on sequence-dependent alterations in the conformation and/or conformational flexibility of the noncontacted bases in the DNA site.Since sequence-dependent DNA structural differences play a role in mediating protein-DNA complex formation, this implies that a protein binds only to a distorted DNA conformation. Noncontacted bases may be envisioned to affect the formation of a protein DNA complex in any of three ways: 1) altering the deformability of DNA; 2) altering the structure of the DNA in the bound complex, thereby changing the strength of particular protein-DNA contacts; or 3) altering the structure of the unbound DNA, thereby eliminating or requiring the imposition of energetically costly large-scale DNA conformational changes. Although indirect readout is part of the sequence recognition mechanism of many sequence-specific DNA binding proteins, little is known about the physical basis of how base sequence and/or the functional groups on the bases contribute to the sequence dependence of such DNA deformations. In addition, the mechanisms by which sequence-dependent differences in DNA conformation influence the strength or specificity of protein-DNA complexes is also unclear.434 repressor does not contact the functional groups on bases at the center of its binding site (1, 2; see also Fig. 1...

Site-specific DNA Transesterification by Vaccinia Topoisomerase

“…4C). The AP:T base pair has two hydrogen bonds and is identical to a G:C base pair in the minor groove but has no hydrogen bond in the major groove, leaving the 4-O of the T without a hydrogen bond donor (41). The AP:T substitution at the Ϫ1 position of the cleavage substrate also had no effect on the rate or extent of transesterification (Fig.…”

“…4A), which results in altered hydrogen bonding properties, both to the opposing pyrimidine base in the complementary strand and, potentially, to proteins that interact with the DNA in the major groove. The AP:C base pair has only one hydrogen bond in the minor groove, leaving the 4-amino group of the C without a hydrogen bond acceptor (41). This modification at the Ϫ1 position of the topoisomerase cleavage substrate had no significant effect on the rate or extent of transesterification (Fig.…”

The Preferred Substrate for RecA-Mediated Cleavage of Bacteriophage 434 Repressor Is the DNA-Bound Dimer

“…All DNAs used in this study were obtained from Integrated DNA Technologies, Inc. (Coralville, Iowa) or through the CAMBI DNA facility (University at Buffalo). DNAs were purified as described previously (22). Each double-stranded DNA binding site was formed from a self-complementary single-stranded DNA (ssDNA) that created a 14 repressor binding sequence or a nonspecific sequence within a hairpin.…”

“…The affinities of wild-type and HMK-tagged 434 repressors were determined by filter binding as described previously (22). Briefly, a 100-bp DNA fragment containing a 434 O R 1 site was 5Ј end labeled by incubating the DNA with 20 Ci of [␥- 32 P]ATP in the presence of T4 polynucleotide kinase (Invitrogen) for 30 min at 37°C in a buffer containing 50 mM Tris (pH 8.0) and 10 mM MgCl 2 .…”

Effect of Salt Shock on Stability of λ imm434 Lysogens

“…Our in vitro results demonstrating that changing the monovalent cation type and concentration differentially affects 434 repressor binding to its various operator sites (32,33) suggested to us that an increased internal salt concentration might affect the interaction of the repressor with DNA, at least transiently. This hypothesis predicted that changing the external salt concentration would influence the stability of imm434 lysogens stabilized by the DNA binding activity of 434 repressor.…”