Migration of a Holliday Junction through a Nucleosome Directed by the E. coli RuvAB Motor Protein  Mikhail Grigoriev, Peggy Hsieh  Molecular Cell  Volume 2, Issue 3, Pages 373-381 (September 1998) DOI: 10.1016/S1097-2765(00)80281-6

Figure 1 Branch Migration of Histone-Free Holliday Junctions by RuvAB (A) In vitro branch migration scheme. 32P-labeled S1 and unlabeled S2 substrates, each having heterologous single-strand tails denoted in white and gray, are rapidly annealed to form a Holliday junction at a defined position. Branch migration of the Holliday junction results in the irreversible formation of two heteroduplex products. Asterisk denotes the 32P label. Throughout the paper, “heteroduplex” denotes the products of branch migration regardless of the extent of sequence homology. (B) Branch migration substrates. (Top) The S1 substrate has a duplex region 250 bp in length, shown in black. The open boxes represent six tandem repeats of a TG octamer positioning sequence shown in the insert. (Bottom) A semimobile S2 substrate containing an 11 bp heterologous insertion at the HincII site (closed box). (C) Branch migration reactions containing semimobile junctions in the presence or absence of RuvA and RuvB proteins were carried out as described in Experimental Procedures and analyzed on native 4.5% polyacrylamide gels. Lanes 1–4: 32P-labeled, naked S1 substrate in the absence (lane 1) or in the presence of RuvA (lane 2), RuvB (lane 3), or RuvAB (lane 4). Lanes 5–11: branch migration of semimobile Holliday junction intermediates in the absence of RuvAB (lane 5); or in the presence of RuvA (lanes 6 and 7), RuvB (lanes 8 and 9), or RuvAB (lanes 10 and 11); or in the absence (lanes 6, 8, and 10) or presence (lanes 7, 9, and 11) of ATP. (D) Scheme for RuvAB-directed branch migration. Only radiolabeled products denoted with asterisks are observed. Molecular Cell 1998 2, 373-381DOI: (10.1016/S1097-2765(00)80281-6)

Figure 2 RuvAB-Directed Branch Migration through a Nucleosome Core (A) A reconstituted S1 substrate showing the major position of the histone octamer indicated by the oval. (B) Branch migration of octamer-reconstituted junction intermediates in the presence of RuvAB. Branch migration assays were carried out for 10 min at 37°C as described in Experimental Procedures with 32P-labeled junction intermediates reconstituted with a histone octamer in the presence or absence of RuvA and RuvB proteins and ATP as indicated and analyzed on 4.5% native polyacrylamide gels. Lanes: 1, 100 bp ladder; 2, histone-free S1 substrate; 3, S1 substrate after histone octamer reconstitution. Lanes 4–10: branch migration reactions of Holliday junction intermediates in the absence (lane 4) or presence of RuvA (lanes 5 and 6), RuvB (lanes 7 and 8), or RuvAB (lanes 9 and 10). (C) The predominant product of branch migration through an octamer is a histone-free heteroduplex. Standard branch migration reactions of histone-free junctions or histone-reconstituted junctions in the presence or absence of RuvAB were analyzed on a 6% native polyacrylamide gel. Lane 1, histone-free S1 substrate; lane 2, spontaneous branch migration reaction involving histone-free junctions after 60 min at 37°C; lane 3, RuvAB-directed branch migration through a histone octamer for 10 min at 37°C. (D) Scheme for RuvAB-directed branch migration through a nucleosome core. Molecular Cell 1998 2, 373-381DOI: (10.1016/S1097-2765(00)80281-6)

Figure 4 Dissociation of the Octamer during Branch Migration (A) Transfer of an octamer to a competitor DNA. Branch migration assays containing histone-reconstituted junctions and RuvAB proteins were carried out in the presence (lanes 6–9) or absence (lanes 2–5) of ATP and a 32P-labeled competitor DNA consisting of a 233 bp fragment containing the TG positioning motif. The molar excess of naked competitor DNA was 10-fold (lanes 2 and 6), 20-fold (lanes 3 and 7), 100-fold (lanes 4 and 8), or 200-fold (lanes 5 and 9). The same 32P-labeled competitor fragment reconstituted with a histone octamer was used as a marker for the products of octamer transfer indicated by the brackets (lane 10). Lane 1 is a 100 bp ladder. (B) Quantification of the extent of octamer transfer. (C) Branch migration by RuvAB at low temperature. Branch migration of histone-free Holliday junction intermediates was carried out in the presence of RuvAB proteins and ATP at 37° (circles), 20° (squares), and 10°C (triangles) as described in Experimental Procedures. (D) Branch migration of histone-reconstituted junction intermediates in the presence of RuvAB proteins and ATP was carried out at 37°C, 20°C, and 10°C as described in Experimental Procedures and analyzed on a native polyacrylamide gel. Molecular Cell 1998 2, 373-381DOI: (10.1016/S1097-2765(00)80281-6)

Figure 3 Kinetics of RuvAB-Directed Branch Migration (A) Branch migration assays utilizing histone-free (top) or octamer-reconstituted (bottom) substrates were carried out as described in Experimental Procedures at 37°C for varying times in the presence or absence or RuvAB proteins and ATP as indicated. Following deproteinization to facilitate quantitation, samples were analyzed on native polyacrylamide gels. Under these electrophoretic conditions, the starting substrate comigrates with heteroduplex products. (B) Comparison of the rate of RuvAB-directed branch migration of histone-free substrates (closed circles) and histone octamer–containing substrates (open circles). The extent of branch migration was determined by quantitating the amount of heteroduplex product formed relative to total labeled DNA and is normalized relative to 80 min of incubation. (C) The time course of branch migration by RuvAB of junction intermediates containing the histone octamer alone (circles), the histone octamer plus H1 (triangles), and the histone octamer plus H5 (squares). Molecular Cell 1998 2, 373-381DOI: (10.1016/S1097-2765(00)80281-6)