Hin Salmonella Invertase Binding to DNA
This tutorial is designed to familiarize one with the characteristics of Hix sites that allow Hin binding. It also shows possible places where the Hix site can be altered in order to create one-time Hix sites.
This view shows the DNA-binding domain of Hin (in spacefill mode) bound to a segment of DNA, colored light blue. Although one half of the Hix site is displayed here along with one Hin protein, the structure in vivo consists of two Hix half sites arranged in an inverted repeat. Hin behaves as a dimer; each Hin binds to one Hix half-site.
The Hin protein in this visualization consists only of the DNA-binding portion of Hin. The actual Hin protein contains a structure away from the DNA that is involved in complexing with other proteins and with cutting the DNA; this segment is not displayed in the Jmol file.
View DNA only
The DNA segment only, in cartoon form.
Color by Nucleotide
|A = Red
T = Green
G = Yellow
C = Blue
Although the bases in this visualization do not correspond completely to the actual bases that bind to Hin, the important DNA structures are present.
|HixC wt half-site
Bases in the visualization
Begin at the top of the diagram - ignore the T that is not bound to a complementary base; it does not exist in the actual HixC site. The first G at the top corresponds to the third base in the half-site, above. Therefore, the actual site where Hin cuts the DNA is out of the visualization. However, Hin never binds to this segment. Continuing down the strand, the rest of the bases in the Hix half-site are present; however, two extra nucleotides (GA) at the bottom of the visualization are not in HixC.
Many of these 13 bases in the HixC site
are involved in binding to the Hin DNA-binding domain. Bases 4, 5, and 6 bind to the N-terminus of Hin through the minor groove of the DNA.
Bases 9, 10, and 11 also bind to Hin through intermediate water molecules and hydrogen bonding in the major groove of the DNA.
Finally, the C-terminus of the Hin DNA-Binding domain complexes with bases 12 and 13 in the minor groove of the DNA.
Based on the data above, I decided to alter HixC in two ways:
First, I would insert a nucleotide inbetween these two bases in the left Hix site, while deleting one of the flanking nucleotides at these points in the right Hix site:
|BP location||Left Hix site||Right Hix Site|
|Between 3 and 4|
When recombined, these site pairings would produce one Hix site that included an insertion and a mutation, altering the relative position of the cut site in relation to Hin and possibly preventing DNA cleavage, leading to a non-functional Hix site after one flip.
Second, Hughes et al. (1992) reports that the following bases, when mutated, display weak Hin-flipping activity:
If two of these sites were paired, recombination would produce Hix sites that had two deleterious mutations, possibly reducing Hin activity significantly and creating one-time Hix sites. .
Feng, Jin-An, Reid C. Johnson, and Richard E. Dickerson. "Hin Recombinase Bound to DNA: The Origin of Specificity in Major and Minor Groove Interactions." Science 263:1994 January 21, 348-355
Hughes, Kelly T. et al. "Sequence-specific interaction of the Salmonella Hin recombinase in both major and minor grooves of DNA. EMBO Journal 11(7):1992, 2695-2705.
Website created by Bruce Henschen, as part of an undergraduate independent study course at Davidson College.