Alternative arrangements of telomeric recognition sites regulate the binding mode of the DNA-binding domain of yeast Rap1

Abstract

The function of yeast Rap1 as an activator in transcription, a repressor at silencer elements, and as a major component of the shelterin-like complex at telomeres requires the known high-affinity and specific interaction of the DNA-binding domain (DBD) with its recognition sequences. In addition to a high-affinity one-to-one complex with its DNA recognition site, Rap1(DBD) also forms lower affinity complexes with higher stoichiometries on DNA. We proposed that this originates from the ability of Rap1(DBD) to access at least two DNA-binding modes. In this work, we show that Rap1(DBD) binds in multiple binding modes to recognition sequences that contain different spacer lengths between the hemi-sites. We also provide evidence that in the singly-ligated complex Rap1(DBD) binds quite differently to these sequences. Rap1(DBD) also binds to a single half-site but does so using the alternative DNA-binding mode where only a single Myb-like domain interacts with DNA. We found that all arrangements of Rap1 sites tested are represented within the telomeric sequence and our data suggest that at telomeres Rap1 might form a nucleoprotein complex with a heterogeneous distribution of bound states.

Keywords: Analytical ultracentrifugation; Fluorescence anisotropy; Protein–DNA interaction; Telomeres.

Figure 1. Rap1^DBD binds to a wide array of telomeric sequence arrangements

(a) Distribution of telomeric sequences that contain a given number of base-pairs separating two tandem repeats. (b) Schematic of the model dsDNA substrate used in this work. (c) EMSA as a function of fold-excess Rap1^DBD for 30 nM TeloS (left) and TeloN (right) labeled at the 5’-end of the top strand with FAM (Table 1). (d) Same experiments as in (c) but at 300 nM DNA.

Figure 2. Rap1^DBD binds to a substrate containing 1 bp spacing between the two half-sites

(a) Change in fluorescence anisotropy as a function of Rap1^DBD/DNA ratio for 255 nM TeloS (circles) or TeloA_h3 (squares) labeled at the 5’-end of the top strand. (b) Same experiments as in (a) but monitoring the change in relative total intensity for the same dsDNAs FAM-labeled at the 3’-end of the top strand.

Figure 3. Rap1^DBD binds to substrates that do not contain any base-pair spacing between the half-sites

(a) Change in fluorescence anisotropy as a function of Rap1^DBD/DNA ratio for 255 nM TeloN labeled with FAM at the 5’-end (black circles) or the 3’-end (black squares) of the top strand. (b) EMSA of Rap1^DBD:DNA complexes formed at a 1:1 ratio with 300 nM DNA substrates containing a different spacing between the half-sites for two total lengths of the DNA.

Figure 4. Rap1^DBD binds to substrates that contain a single half-site

(a) EMSA as a function of fold-excess Rap1^DBD for 300 nM HEMI (left) and HEMI-L (right) labeled at the 5’-end of the top strand with FAM (Table 1). (b) Same experiments as in (a) but performed with 1 μM DNAs. (c) Change in fluorescence anisotropy as a function of Rap1^DBD/DNA ratio for 255 nM HEMI (black circles), HEMI-L (gray circles) and TeloS_h1 (black squares) FAM-labeled at the 5’-end of the top strand. (d) Change in relative total intensity as a function of Rap1^DBD/DNA ratio for 255 nM HEMI (black circles), HEMI-R (gray circles) and TeloS_h1 (black squares) FAM-labeled at the 3’-end of the top strand. (e) Sedimentation coefficient distributions from velocity experiments of 2 μM Cy3-labeled HEMI (black) and TeloS_h1 (gray) in Buffer HN₅₀ at equimolar Rap1^DBD/DNA loading ratio. For clarity the region corresponding to free DNA has been excluded.

Figure 5. At telomeres Rap1 can form a nucleoprotein complex with heterogeneous distribution of bound states

Cartoon model of a possible distribution of Rap1 bound states at different binding sites with variable spacing between the half-site, 3 bp (black), 1 bp (blue), none (green) or isolated half-site (gray).