Oligonucleotides were purchased from Eurogentec, purified by denaturing gel electrophoresis

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1 SUPPLEMENRY INFORMION Purification of probes and Oligonucleotides sequence Oligonucleotides were purchased from Eurogentec, purified by denaturing gel electrophoresis and recovered by electroelution. Labelling was performed using γ 32 P P and 4 PNK (Promega). ll probes were prepared by incubation at 85 in 2 mm ris-l ph8, 5 mm Nal, followed by slow cooling overnight followed by purification by native gel electrophoresis. he sequences are as follows: nsphj Strand 1 5 Strand 2 5 Strand 3 5 Strand 4 5 ds2: strand 1 from thj and the following sequence 5 ds4 Strand1 5 Strand 2 5 ds2: strand 1 from thj and the following sequence 5 dsl2 : strand 1 from thj and the following sequence

2 5 18ds2: 5 5 S1: S1 top strand 5 S1 bottom strand 5 S2: S2 top strand 5 S2 bottom strand 5 hs2: hs2 top strand 5 hs2 bottom strand 5

3 Peptides sequence peptide MSSD SRRRS RSSRRRR HEPLPE RE SRRRS RSSRRRR HE Histidine 31 is underlined

4 Strand 1 5 Strand 2 5 Strand 3 5 Strand Supplementary Figure 1 he telomeric HJ is constructed by hybridization of four 54 nt oligonucleotides. () Sequence of the four 54 nt oligonucleotides used for the construction of thj. () omparative migration on acrylamide gel: strand 1 (lane 1), species obtained after hybridization of strands 1 and 2 (lane 2) or strands 1, 2 and 3 (lane 3), the four purified thjs labelled on strand 1, 2, 3 or 4 (lanes 4 to 7 respectively) and a 18 bp double stranded probe as size marker (lane 8).

5 M D L M nm nm 1 M nm Supplementary Figure 2 he domain is essential for efficient binding of on a telomeric HJ. () Schematic drawing of the thj center showing the telomeric homologous region (boxed sequence) and 3 of the 13 possible positions of the junction branch point (numbered from -6 to +6). () Schematic drawings of the different mutant and wild type forms of, letters above indicate the different domains of and numbers the starting and ending positions of each domain. stands for N-terminal basic domain, D for dimerization domain, L for linker domain and M for Myb-like domain. () EMSs performed on labelled thj (5 nm) using 1, 2, 3, 5, 75 and 1 nm of either, or M. Lanes 1 show the junction in the absence of protein.

6 thj nsphj ds2 free thj free thj free thj M fb/fb fb/fb fb/fb M [comp]/[thj] [comp]/[thj] [comp]/[thj] vs vs vs thj thj thj nsphj thj ds2 Supplementary Figure 3 he domain of exhibits a marked preference for the Holliday junction structure. () ompetition experiments: 5 nm of labelled thj was incubated with 5 nm of (top gel), 2 nm of (middle gel) or 5 nm M (bottom gel) in the presence of increasing amounts of cold competitor, thj, nsphj (a semi-mobile junction containing a scrambled sequence of the telomeric repeat) and ds2 (a double stranded probe containing two telomeric repeats). he concentrations of competitors were, 5, 6.25, 7.5, 1, 15 and 25 nm (lanes 1 to 7 respectively). () raph representing the variations of the ratio of the DN bound fractions (fraction bound in the sample with competitor, fb, over fraction bound in the sample without competitor, f) as a function of the ratio between the concentrations of labelled thj and the cold thj (closed squares), cold nsphj (open squares) and cold ds2 (open triangles) competitors.

7 strand 1 strand 2 uncleaved EN1 + uncleaved EN1 + uncleaved strand 3 EN1 + uncleaved strand 4 EN Supplementary Figure 4 EN cleaves a telomeric HJ in a sequence-dependent manner. () EN cleavage products obtained after incubation of thj labelled on either of the four strands, the cleavage pattern is compared to that obtained following a Maxam and ilbert sequencing (+) reaction on the corresponding strands. On can observe the preferential cleavages in the guanine tracts of strands 1 and 3. () leavage sites positions on thj, arrows and asterisks represent the major and minor cleavage sites respectively.

8 strand 2 EN1 EN1 + EN1 + EN1 + M M strand 3 EN1 EN1 + EN1 + EN1 + M M Supplementary Figure 5 he domain of controls protection while the Myb-like domain is responsible for the 5' side activation. () 5 nm of thj labelled on strand 2 was incubated with 2 nm of, or M prior to cleavage with EN he first lane shows the uncleaved junction and in the three last lanes only telomeric proteins were added. Numbers below the gel represent the variations of intensities of cleavage in % () in the samples containing the telomeric proteins compared to the sample only containing the enzyme. Negative numbers show protection, positive numbers activation of cleavage. Note the apparent resistance of the 5' located cleavage site ( ) to -mediated inhibition. () Same experiment as in () with strand 3.

9 M M strand 1 7 Endo I 7 Endo I cleavage profile Endo I [] nm Endo I [ ] nm D [ M] nm Supplementary Figure 6 2 Endo I + M

10 Supplementary Figure 6 protects thj from cleavage by 7 Endo I in a domain-dependent manner. () leavage experiment performed on 5 nm of thj labelled on strand 1 in the presence of increasing amounts of, or M. oncentrations used were 1 nm, 2 nm, 5 nm, 1 nm and 2 nm. Lane 1 shows undigested strand 1 and lane 2, background 7 Endo I cleavage. In lanes 18-2 only 2 nm of telomeric proteins were added. Positions of cleavage sites were assigned by comparison to a Maxam and ilbert sequencing reaction of the strand (not shown). On the left side is presented the cleavage profile obtained in the absence of telomeric protein and the position of each cleavage. () Left panel: graph representing the variations in % of the intensities of each cleavage band as a function of concentration. he lines correspond to average values calculated for bands exhibiting similar behaviour. hree behaviours could be observed: some band intensities were not altered (open symbols), some were increased (grey symbols) or decreased (black symbols). Middle panel: comparison of cleavage profiles for 7 Endo I alone (grey line) or in the presence of 2 nm of (black line). he symbols above the profiles are the ones used in the left panel and each correspond to the peak below them. Right panel: data obtained for all strands are summarized, empty boxes corresponding to unaltered positions, grey boxes to sites of increased cleavage and black boxes to sites of decreased cleavage. () Same as () for. (D) Same as () for M.

11 Strand 2 Endo I Β Μ Β Μ Strand 3 Endo I Β Μ Β Μ Strand 4 Endo I Β Μ Β Μ [] nm [] nm [ R F 2] nm [ ] nm [ ] nm [ R F 2 ] nm [ M ] nm [ M ] nm [ M ] nm Supplementary Figure 7

12 Supplementary Figure 7 protects thj from 7 Endo I cleavage in a domain-dependent manner. () top: cleavage experiment performed with 5 nm of thj labelled on strand 2 in the presence of increasing amounts of, and M. oncentrations of telomeric proteins used were 1, 2, 5, 1 and 2 nm. he first lane shows undigested strand 2 and the second 7 Endo I cleavage. In the last three lanes 2 nm of telomeric proteins were added alone. op graph: graph representing the variations of the intensities of each cleavage band () as a function of concentration. he lines were drawn using average values for bands exhibiting the same behaviour. Open symbols correspond to invariant intensities, grey symbols to increasing intensities and black symbols to decreasing intensities. Middle graphe: same graph as above for. ottom graph: same graph for M. () and () correspond to the same experiment as in () performed on strands 3 and 4 respectively.

13 E E1 cleavage profile strand 4 MP-E1 Rus cleavage profile strand 4 MP-Rus strand 4 MP-E1 + + M MP-Rus + + M M D F E1 + + Rus + + H 1 E1 + + M I 1 Rus + + M [protein] nm [protein] nm Supplementary Figure 8

14 Supplementary Figure 8 protects thj from cleavage by E1 and Rus in a -dependent manner. () MP-E1 cleavage experiment performed with 5 nm of thj labelled on strand 4 in the presence of increasing amounts of and. oncentrations of telomeric proteins used were 1, 2, 5, 1 and 2 nm. he first lane shows undigested strand 4 and the second lane MP-E1 cleavage. In lanes 13 and 14 only 2 nm of telomeric proteins were added. () omparison of cleavage profiles for MP-E1 alone (grey line) or in the presence of 2 nm of (black line) or (dotted line). () and (D) Same experiments as in () and () for MP-Rus. (E) MP-E1 and MP-Rus cleavage experiments performed with 5 nm of thj labelled on strand 4 in the absence or presence of 2 nm of or M. he first lane shows undigested strand 4 and lanes 2 and 5 MP-E1 and MP-Rus cleavage respectively. In lanes 8 and 9 only 2 nm of telomeric proteins were added. (F) omparison of cleavage profiles for MP-E1 alone (grey line) or in the presence of 2 nm of (black line) or of M (dotted line). () Same comparison as in (F) for MP-Rus cleavage. (H) raph representing the variations in % of MP-E1 cleavage intensity () as a function of the concentration of (closed squares) or (open squares). Values were obtained by averaging those obtained for the two major cleavage bands since they exhibited the same behaviour. Error bars correspond to the maxima and minima obtained for these values. (I) Same as (H) for MP-Rus.

15 strand 4 7 Endo I RF1 RF1 strand 4 MP-ceI MP-ceI + RF1 MP-Rus MP-Rus + RF1 RF1 Endo I cleavage pattern cei cleavage profile Rus cleavage profile 5' 5' 5' D Endo I +RF1 E E1 +RF1 Supplementary Figure 9 RF1 does not protect against Endo I, cei and Rus cleavage () Endo I cleavage experiment performed with 5 nm of thj labelled on strand 4 in the presence of increasing amounts of RF1 (lanes 3 to 7). oncentrations of RF1 used were 1, 2, 5, 1 and 2 nm. he first lane shows undigested strand 4, the second Endo I cleavage and in lane 8 only 2 nm of RF1 was added. Note the activation of cleavage obtained for the 5' located cleavage sites ( ). () omparison of cleavage profiles for Endo I alone (light grey line) or in the presence of 2 nm of RF1 (dark grey line). () MP-ceI and MP-Rus cleavage experiments performed with 5 nm of thj labelled on strand 4 in the presence of 2 nm of RF1. he first lane shows undigested strand 4 and in lane 6 only 2 nm of RF1 was added. (D) and (E) omparison of cleavage profiles for MP-ceI or MP-Rus alone (light grey lines) and in the presence of 2 nm of RF1 (dotted lines). Rus +RF1

16 Substrates Junction (23)4 (23)4 + S1 S2 annealing migration dissociation Products P1 P2 S1 S1+S2 S1 H H P1 H H Junction S1 P1 S1 1 2 P D Substrates (23)4 (23)4 + S1 hs2 annealing Junction E S1 S1+S2 S1+hS2 Junction S1 P1 Supplementary Figure 1

17 Supplementary Figure 1 he migration assay. () Reaction scheme of the migration assay. () Electrophoresic migration of the S1 substrate (lane 1) and the sample obtained after 6 hours incubation (lane 2). () Electrophoretic migration of the S1 substrate (lane 1), amhi digested (lane 2) or HindIII digested (lane 3) S1 substrate, P1 product (lane 4), amhi digested (lane 5) or HindIII digested (lane 6) P1 product. and H marks show the positions of the amhi and HindIII restriction sites respectively. (D) Reaction scheme of the migration assay performed in the presence of the hs2 substrate which stops migration. (E) Electrophoretic migration of the S1 substrate (lane 1), outcome of the assay after 6 hours incubation using the S2 substrate (lane 2) or the hs2 substrate (lane 3).

18 Junction minutes % Junction k1 > 1 7 M -1.s -1 P k2 = 2 ± s -1 S1 P time (min) Supplementary Figure 11 greatly increases the junction formation but slows down its migration. () Migration assay performed in the presence of 1 nm of, aliquots were taken at long time points. () Variations of the percentage of the S1 substrate, junction and P1 product through time corresponding to the experiment shown in (). he lines represent the fitting obtained with the rates noted inside the figure which were calculated using the experimental data shown in Figure 3. One can note the good fitting of the experimental data with the parameters obtained with an independent shorter experiment.

19 RF nm wells free DN Supplementary Figure 12 gives complexes similar to those of the dimeric RF1 with a double stranded telomeric probe. crylamide gel electrophoresis of samples obtained by incubation of 5 nm of labelled ds4 probe with increasing amounts of (lanes 2 to 4), (lanes 5 to 7) or RF1 (lanes 8 to 1). oncentrations used are indicated above the gel.

20 M - peptide RF1 KMnO 4 M peptide RF R F 2 R F 2 R F 2 M peptide R F 1 free thj free thj free thj Strand 1 Strand 2 Strand Strand 4 free thj KMnO 4 M peptide RF R F 2 R F 2 R F 2 M peptide R F 1 D KMnO 4 M peptide RF R F 2 R F 2 R F 2 M peptide R F Supplementary Figure 13

21 Supplementary Figure 13 Permanganate probing of thj on strands 2 to 4. () EMSs on thj labelled on strand 1 (top panel), strand 2 (middle-top panel), strand 3 (middle-bottom panel), strand 4 (bottom panel) in the presence of 5 nm of either RF1,, or M and 5 µm of the peptide. () permanganate probing experiment performed with 5 nm of thj labelled on strand 2 in the presence of 5 nm of either RF1,, or M and 5 µm of the peptide. Lane 1 shows undigested strand 2. Lane 2 and 3 show the background cleavage obtained through piperidine cleavage alone and with permanganate respectively. Sequence of the telomeric tract is given on the left side. On the right side are presented the relative intensity profiles obtained after quantification of the sample lanes. () and (D) same experiments as in () with strands 3 and 4 respectively.

22 I/I pep 4, 41 and 42 4 M 4, 41 and and 42 4, 41 and [] or [ ] nm [ M ] nm [ pep] nm Supplementary Figure 14 Quantitative analysis of permanganate hypersensitivity as a function of proteins concentration on strand 4. () Schematic representation of thj, the boxed sequence corresponds to the homologous telomeric core in strand 4. () raph showing the stimulation of permanganate reactivity (ratio between the intensity of the corresponding bands with, I, and without added protein, I) on thymines 4, 41 and 42 of strand 4 in the presence of increasing concentration of wild type and various mutants of. For, M and the peptide the values obtained for the three thymines were averaged since they exhibited the same behaviour. For wild type, 4 reactivity was different from that of 41 and 42 and was therefore considered separately. Errors bars correspond to standard deviations between values for the corresponding thymines in different sets of experiments. One can note the weak response obtained for and the reactivity of thymines in the presence of wild type, M and the peptide. he same experiment performed on the other three strands gave similar results (data not shown).

23 dsnsp dsl2 ds dsnsp vs dsnsp dsnsp vs dsl dsnsp vs ds dsnps ds2 dsl2 f/f [competitor]/[dsnsp] Supplementary Figure 15 M preferentially binds to a 2 bulge. () Left panel: agarose gel of the competition experiment performed using 5 nm of the labelled dsnsp probe, 2 nm of M and increasing concentrations of cold dsnsp probe. Middle panel same experiment as in the left panel with the 2 bubble competitor (dsl2). Right panel same experiment as in the left panel with the 2 bulge competitor (ds2). () raph representing the variations of the ratio of the DN bound fractions (fraction bound in the sample with competitor, fb, over the fraction bound in the sample without competitor, f) as a function of the ratio between the concentrations of labelled dsnsp and cold dsnsp (open diamonds), cold dsl2 (grey triangles) and cold ds2 (closed triangles) competitors.

24 strand 1 EN1 EN1 + H- H- strand 1 Endo I H- H- strand 2 Endo I H- MP-E I H- H D strand 3 Endo I H- H- E strand 4 Endo I H- MP-E I H- H Supplementary Figure 16

25 Supplementary Figure 16 he H- mutant does not protect thj from cleavage by EN , 7 Endo I and cei. () EN cleavage experiment performed on 5 nm of thj labelled on strand 1 in the absence (lane 2) or the presence of 2 nm of H- (lane 3). Lane 1 shows undigested strand 1 and in lane 4 only 2 nm of H- was added. () 7 Endo I cleavage experiment performed on 5 nm of thj labelled on strand 1 in the presence of 2 nm of or increasing amounts of H-. oncentrations of H- used were 5, 1, 2, 5 and 1 nm. he first lane shows undigested strand 2. In the last two lanes the highest concentration of each telomeric protein was added alone. () Same experiments as in () on strand 2 using 7 Endo I and MP-ceI (D) Same experiment as in () on strand 3. (E) Same experiment as in () on strand 4.