Creación de la secuencia didáctica

CHRAC14-CHRAC16 bind to DNA in a non-specific manner. As a consequence, more than one heterodimer can bind to the same DNA double helix if the fragment is long enough. This binding behaviour results in indistinct, blurred bands in EMSAs, especially if the DNA binding is not very strong and the protein-DNA complex dissociates during gel electrophoresis. Indeed, this can be observed in Figure 4.19.

Therefore, the dependence of p14-p16 DNA binding on DNA fragment length was tested in a DNA pull-down assay. For this analysis, full length p14-p16 and p14-p16ΔC were

immobilised on sepharose beads and incubated with a radiolabelled 10 base pair DNA ladder. Bead-bound DNA and DNA in the supernatant was separated by denaturing gel- electrophoresis and quantified (see 3.2.19, Figure 4.20 A). The data for the full length heterodimer were hard to interpret, because the portion of DNA that remained bound to the beads was below 3% for each fragment within the analysed range of DNA fragments between 10 and 100 bp (Figure 4.20 B).

However, both the full length and the p14-p16ΔC heterodimer bound only traces of the ten base pair DNA fragment (Figure 4.20 A lanes 11 to 14 and Figure 4.20 B, C). These results suggest that the minimal DNA fragment length required for p14-p16 binding is more than ten base pairs. This is consistent with the model of CHRAC14-CHRAC16 DNA binding derived from the nucleosome structure (Figure 4.18). According to this model, approximately 30 DNA base pairs span the surface of the p14-p16 histone fold core.

For the p14-p16ΔC deletion variant, the amount of interacting DNA increased in the range between 30 and 70 base pairs, until it reached a plateau and no further increase was observed (Figure 4.20 C). However, the supernatant was depleted to a great extent of DNA fragments larger than 70 base pairs in these experiments (see Figure 4.20 A lanes 7/8 and Figure 4.20 C), and therefore it is likely that the interaction with DNA still increases with DNA length for fragments larger than 70 base pairs.

Figure 4.20: Determination of the length-dependence of p14-p16 DNA binding. A: Autoradiography of a DNA pull-down assay. Input (lanes 1 and 2), supernatant (lanes 3 to 8) and beads (lanes 9 to 14) are analysed on a 8 % denaturing gel. Mock: TALON beads only (10 µL, lanes 3/4 and 9/10). Total amount of protein on TALON beads: 200 pmol (lanes 5/11 and 7/13, respectively) and 100 pmol (lanes 6/12 and 8/14, respectively). B: Quantification of DNA pull-down with 200 pmol p14-p16. Unbound fraction: supernatant, lane 5 in panel A; bound fraction: beads, lane 11 in panel A. C: Quantification of DNA pull-down with 200

pmol p14-p16ΔC. Unbound fraction: supernatant, lane 7 in panel A; bound fraction: beads, lane 13 in panel A.

The electrophoretic mobility shift assay shown in Figure 4.21 argues for such a scenario. Full length CHRAC14-CHRAC16 does not shift the 35 base pair DNA fragment (lanes 2-6), and the 72 base pair fragment (lanes 8-12) is shifted only at relatively high protein concentrations (>4 µM), whereas the 248 base pair fragment (lanes 14-18) is shifted already at lower protein concentrations of 1 µM. The same trend can be seen for the CHRAC14- CHRAC16ΔC heterodimer, although the overall DNA binding of this deletion variant is increased and it shifts already the 35 base pair DNA fragment (lanes 20 to 24).

Figure 4.21: DNA-binding of p14-p16 is dependent on DNA length. EMSA of 0.5 nM radiolabelled (end- labelled) DNA with fragment sizes of 35 bp, 72 bp and 248 bp, respectively. Protein concentrations used were

10, 4, 1, 0.4 and 0.1 µM for both full length p14-p16 and p14-p16_ΔC. Full length p14-p16 is unable to shift

the 35 bp DNA fragment under the assay conditions (lanes 2 to 6). Bandshifts with distinct shifted species are only observed with 72 bp DNA (lanes 9/10 and 28 to 30, respectively).

The strength of protein-DNA interactions is often given as a dissociation constant (KD).

The KD can be determined by the Langmuir model if the interaction can be described as a

simple 1:1 complex formation. The binding of more than one protein (or protein complex) to the same DNA fragment or allosteric effects are not taken into consideration by the Langmuir model.

Hence, it is not trivial to determine an accurate KD value for p14-p16 DNA binding.

However, for short DNA fragments it can be assumed that there is only one p14-p16 binding site per DNA fragment and therefore it is feasible to make use of the Langmuir model and

determine an approximate KD value. As the full length CHRAC14-CHRAC16 heterodimer did

not bind to 35 base pair DNA in the electrophoretic mobility shift assay (Figure 4.21), the KD

values for both the full length heterodimer and the p14-p16ΔC heterodimer were determined for the 72 base pair DNA fragment in four independent experiments like the one shown in Figure 4.22 A. The DNA binding could be approximated by a hyperbolic Langmuir curve

(Figure 4.22 B, C). The KD value for full length CHRAC14-CHRAC16 was 2.3 µM, reflecting

its weak DNA affinity, and the K_D value for CHRAC14-CHRAC16ΔC was 57 nM. This

means that the deletion variant binds about 40 times stronger to DNA than the full length heterodimer.

Figure 4.22: A: EMSA with fine titrations of full length p14-p16 and p14-p16_ΔC to 0.5 nM radiolabelled 72 bp DNA for determination of their DNA binding constants. Protein concentrations were 15, 12.5, 10, 7.5, 5.5, 4.5, 3.5, 2.5, 1.5 and 0.5 µM for the full length heterodimer and 2, 1.5, 1, 0.75, 0.5, 0.3, 0.1, 0.04, 0.01 and 0.004 µM for the heterodimer with the C-terminal deletion of p16.

B: Quantification of the electrophoretic mobility shifts caused by full length p14-p16 and determination of the approximate DNA binding constant. In four independent experiments such as the one shown in panel A, the percentage of unshifted DNA was determined using AIDA software (Raytest). The non-linear curve fit was performed with the programme Prism (GraphPad).

C: Quantification of the electrophoretic mobility shifts caused by p14-p16ΔC and determination of the

approximate DNA binding constant. The data analysis was performed as described for panel B.