Y-Structure DNA

We were also interested in Y-structure DNA. This structure was designed by using the partial junction, JunctionB, and adding a complementary sequence to coordinate with the tails. Two sequences were ordered, one which was a perfect complement to the tails (Y-Structure) and one which contained an extra thymidine at the base of the stem loop to give added flexibility (Y-Structure + 1NT) (Figure 3.5). Both structures were not very stable. However, the extra thymidine caused T7 Endo I to cleave this structure to a greater extent than the Y-Structure without it (Figure 3.20). Y-structures, similar to the partial junctions of the last section are also cleaved primarily one nucleotide from the branch point on the 5’ side.

Figure 3.5- Model of the Y-Structure DNA. This diagram shows the two Y-structures we worked with. This model represents the two Y-structures we analyzed, Y-Structure where the red x at the center of the junction is missing and Y-Structure + 1NT, in which the red x is a thymidine.

3.2 Conformational Dynamics

3.2.1 Conformational Effects on Circular Dichroism

It has been shown that Holliday junctions can exist in two different conformations, the Open-X and the Stacked-X forms. It was believed that the difference between the two conformations could be detected with circular dichroism. The Stacked-X conformation has been shown to be the predominant form in the presence of MgCl2. Several spectra were run with MgCl2 being titrated into a solution with

Figure 3.6- Open-X to Stacked-X Conformational Switch. This diagram shows the possible

Figure 3.7- Effect of MgCl2 on the CD spectra of Cruci3HL. CD spectra overlayed of Cruci3HL with MgCl2

titrated in from 0 mM – 12 mM. There is a significant drop in the molar elipticity at 285 nm when MgCl2

is added. 1 OD/mL Cruci3HL, 10 mM NaCl, 10 mM Tris-HCl pH 7.0.

The first addition of MgCl2 drastically changes the CD spectrum by causing a decrease in the

molar elipticity at 285 nm and at 250 nm. Further additions of MgCl2 change the spectra to a lesser

extent. Finally, the sample was heated to 95 °C for 5 minutes and returned to room temperature; this had no effect on the spectrum. Under all ionic conditions studied the cruciform exhibited a B-DNA form. To see if the effect of MgCl2 on the CD spectra was unique to Cruci3HL, a hairpin loop with the same

sequence of an arm of Cruci3HL was run with the same conditions (Figure 3.8). The spectra of the hairpin and of Cruci3HL were almost identical. We can conclude that MgCl2 changes the structure of

Figure 3.8- Effect of MgCl2 on the CD spectra of Cruci3HL-Hairpin-Loop. CD spectra overlayed of Cruci3HL-Hairpin-Loop with MgCl2 titrated in from 0 mM – 12 mM. The spectrum shows the same large

Figure 3.9- Effect of MgCl2 on the CD spectra of Junction3. CD spectra overlayed of Junction3 with MgCl2 titrated in from 0 mM – 16 mM. The spectra looks similar to Cruci3HL and Cruci3HL-Hairpin-Loop

with the dip at 285 nm being less significant. 1 OD/mL Junction3, 20 mM NaCl, 10 mM Tris-HCl (pH 7.0). The CD spectra of Junction3 maintain similarities to Cruci3HL and Cruci3HL-Hairpin-Loop such as the dip in molar elipticity at 285 nm and 245 nm as MgCl2 is titrated into the cuvette. However, the

change in elipticity is much lower for Junction3 at 285 nm. It remains a challenge to determine exactly why this spectra is different, but a possible explanation is that the cruciform formed from Junction3 (JunctionA + JunctionB) is not entirely stable without the presence of MgCl2 as evidenced on several

native gels. With just NaCl present, Junction3 is approximately 90% cruciform and 10% duplex. With the addition of MgCl2 the junction is completely stabilized with no duplex formation (data not shown). This

3.2.2 Conformational Effects on Gel Mobility

Duckett et al. showed that Junction3 will form the Stacked-X or Open-X based on the ionic conditions present via comparative gel electrophoresis[25]_{. Adding 1 mM MgCl}

2 changed the overall

conformation from Open-X to Stacked-X[25]_{. It is expected that the different conformations would have}

different electrophoretic mobilities. A Native PAGE was used to test for this, each gel had Cruci3HL and was flanked by a 90-mer control and a 50-mer control. One gel contained MgCl2 in the running and

loading buffer while the other gel had EDTA in each. The gels show that Cruc3HL runs at the same mobility regardless of the presence of MgCl2. This was determined by measuring the ratios of the

distance of Cruci3HL to the 90-mer and to the 50-mer, the ratios are essentially the same. Again, this doesn’t prove or confirm that MgCl2 causes a conformational change. Continuing on the gel mobility

study which has shown us that Cruci3HL is unaffected by salt changes, we can now use this as a control for different cruciform structures such as Junction3. Junction3’s mobility is significantly impacted by the presence of 2 mM MgCl2 (Figure 3.11). Junction3 travels 16% faster in the presence of MgCl2 compared

Figure 3.10- Conformation Dynamics of Cruci3HL Compared to Single Stranded DNA. This gel compares the effect of MgCl2 on electrophoretic mobility. The lanes are the same in each gel with the exception of

the running buffer, on the left each sample is in a 89 mM Tris-borate buffer containing 5mM MgCl2. The

gel on the right has each sample in a 89 mM Tris-borate buffer with 2 mM EDTA. Lanes 1-3 are 90-mer, Cruci3HL, and the 50-mer respectively.

Figure 3.11- Conformational Dynamics of Cruci3HL compared to Junction3. The gel on the left shows the mobility of Cruci3HL compared to the mobility of Junction3 in the presence of 2 mM MgCl2. Since

Cruci3HL is not affected by salt changes it is used as a reference for the relative mobility of Junction3. The gel on the right has the same conditions but without MgCl2 and in the presence of 2 mM MgCl2.