Mendel-Brno 2000

category image Volume: 17
Issue Number 6, Part 2
June 2000

Sequence Determinants of DNA Structure and the Mechanism of DNA Structure Recognition by 434 Repressor

In complexes of the bacteriophage 434 binding sites with 434 repressor, the central four base pairs of the 14 base pair site are not contacted by the protein, although changes in these bases alter the binding site's affinity for repressor. Our previous data suggested that the ability of the noncontacted central bases to be overtwisted in repressor-DNA complexes governs the affinity of the binding site for 434 repressor. We know that 434 repressor prefers to bind operators bearing A/T bases at their centers over ones bearing G/C base pairs at their central sequences.

One model to explain these observations suggests that the resistance of H-bonds between bases in the twisted regions to be distorted modulates the twisting flexibility DNA. Alternatively, the absence or presence of the N2 group in the minor groove at the center of the binding site may influence protein affinity for DNA. These ideas were tested by examining the affinity of 434 repressor synthetic DNA binding sites that bear non-natural bases at one or more of the central four positions. We find that protein affinity for DNA is independent of the number of hydrogen bonds between the noncontacted base pairs. We also find that in the presence the N2 group on purine bases at the center of the binding site decreases the affinity of repressor for DNA, regardless of sequence context or the identity of the other substituents on the purine base. The N2 group-dependent decrease in affinity is always accompanied by changes in the physical properties of the binding site DNA, i.e., a >/=4.5ยบ decrease in the Tm of the DNA, an increase in the positive CD transition at ~275 nm and an increase in DNase I cleavage at the binding site center. Consistent with the idea that sequence-dependent differences in DNA structure have a role in determining the affinity of DNA for repressor, these latter findings suggest that the N2 group affect protein affinity for DNA by altering an intrinsic property and not by directly modifying protein-DNA interactions.

To gain further insight into how DNA sequence determines DNA structure, in vitro selection experiments were conducted in an effort to uncover the sequence determinants that govern DNA twist and/or twisting flexibility. Using a ring closure assay, we selected DNA sequences from a randomized pool of sequences that enhance the formation of either overwound or underwound DNA topoisomers. Consistent with their higher average twist, the CD intensity of the overwound DNA population is greater than that of the underwound DNAs. Surprisingly, we find that the population of underwound DNAs have substantially higher stabilities than the overwound DNAs, despite the fact that the G/C content of the population of underwound DNAs is lower than that of the overwound DNA population. This observation reemphasizes the importance of sequence in determining stability. Efforts to extract information regarding the sequence determinants that confer over- or undertwisting are ongoing and will be discussed.

Gerald B. Koudelka, Steven Mauro, David R. Pawlowski, Mihai Ciubotaru,

Department of Biological Sciences,
State University of New York at Buffalo,
Buffalo, NY 14260