1. Mechanical Constraints on Hin Subunit Rotation Imposed by the Fis/Enhancer System and DNA Supercoiling during Site-Specific Recombination 
Gautam Dhar, John K. Heiss, Reid C. Johnson 
Molecular Cell 
Volume 34, Issue 6, Pages (June 2009)
DOI: /j.molcel
Copyright © 2009 Elsevier Inc. Terms and Conditions

2. Figure 1
Crosslinking of Invertasomes Assembled during Hin-Catalyzed Site-Specific Recombination
(A) Pathway of the Hin-catalyzed site-specific DNA inversion reaction. Recombination proceeds by a single clockwise rotation of the top pair (yellow and purple) of Hin subunits, which are covalently linked to cleaved DNA ends, to form new dimers with exchanged DNA strands.
(B) Structural model of the synaptic Hin tetramer based on the crystal structure of γδ resolvase. The view highlights the “flat interface” whereby the top pair (yellow and purple) of synapsed subunits can rotate relative to the bottom pair (green and blue). Residues discussed in this paper that support efficient crosslinking are labeled.
(C) Subunit rotation mechanism. Hin tetramer model in the orientation as in (B) (i) and after a 90° rotation about the x axis (ii). The long E helices from each subunit are highlighted. Cys134, orange spheres; Cys94, red spheres; Cys101, gray spheres. A 90° clockwise (iii) or counterclockwise (iv) rotation of the yellow and purple subunits generates helix E-aligned conformers and positions pairs of cysteines for crosslinking. Additional 90° rotations generate the recombinant DNA-aligned conformation (v).
(D) Strategy used for cysteine-dependent crosslinking and 32P end labeling of Hin-DNA covalent complexes formed on supercoiled DNA plasmids (see Figure S1 for details on the plasmid structures). Crosslinks between different pairs of subunits are depicted.
(E) Crosslinking of Hin-DNA complexes by Hin cysteine mutants. Fis-activated invertasomes were assembled on pRJ2372 for 20 min and crosslinked for 1 min with diamide (0 Å), BMOE (8 Å), BMH (16 Å), or a no crosslinker control (none); no Hin was added to the reaction labeled “-Hin.” The fastest migrating species is the monomeric Hin-(32P)54 nt covalent complex, and the slower migrating species is the crosslinked dimeric Hin-(32P)54 nt product. An additional product present with Hin-S94C, D96C, S99C (trace), and M101C (trace) are crosslinked dimers in which only one Hin subunit is covalently associated with DNA. Plus sign (+) indicates the location of a minor Hin-DNA cleavage band that appears in different amounts independent of crosslinking and asterisk (∗) indicates the location of a DNA-only band that is derived from the invertible segment.
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3. Figure 2
Crosslinking between Cysteines Located at the C-Terminal End of Helix E
(A) Schematic representation of the Hin tetramer undergoing clockwise or counterclockwise subunit rotations to generate Cys134 crosslinks (see Movies S1A and S1B). The cartoons are modeled after Figure 1C (ii–v) with the catalytic and DNA binding domains represented by large and small ovals separated by the E helices bound to different length DNA segments generated after EcoRI digestion. Red stars denote the locations of Q134C on each subunit and the boxed cartoons depict the crosslinked products resolved by SDS-PAGE.
(B) Fis-activated Hin-Q134C crosslinking reactions on supercoiled pRJ2330. Crosslinking was for 20 s with BMOE (8 Å spacer) at the designated times after Hin addition. The portion of the SDS-polyacrylamide gel showing the crosslinked products is shown (see Figure S2A for the autoradiograph of the full gel). The predominant crosslinked hetero-diprotomer species containing the Hin-54 nt (32P-labeled) and Hin-18 nt (unlabeled) migrates fastest, the crosslinked homo-diprotomer containing labeled 54 nt segments migrates near the top, and the crosslinked homo-diprotomer containing unlabeled 18 nt segments is not visible in the autoradiograph. Control lanes on the right show the crosslinked nt homo-diprotomer from reactions performed on pRJ2372, a no crosslinker control, and a no Hin control. The asterisk marks a vector DNA band from the invertible segment.
(C) Crosslinking reactions using the Fis-independent mutant Hin-H107Y/Q134C on open-circular pRJ2330.
(D) Plot of the relative proportion of the nt hetero-diprotomer crosslinked products with respect to Hin incubation time obtained from quantifying the results of (B) and (C).
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 Crosslinking between Cysteines Located at Residue 94
(A) Model of the Hin tetramer undergoing clockwise or counterclockwise subunit rotations to generate Cys94 crosslinks (see Movies S2A and S2B). The outlay is similar to Figure 2A. Although the helix E-aligned rotational conformers are depicted here, clockwise rotations of 90°–155° and counterclockwise rotations of 25°–90° are predicted to support crosslinking (Figure S6B).
(B) Fis-activated Hin-S94C crosslinking reactions on supercoiled pRJ2330 performed as in Figure 2B.
(C) Fis-independent Hin-H107Y/S94C crosslinking reactions on open-circular pRJ2330.
(D) Plot of hetero-diprotomer crosslinked products with respect to Hin incubation time. Data from Hin-H107Y/S94C crosslinking reactions on 112 bp and 40 bp hix fragments (Figure S4A) are also included.
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5. Figure 4 Crosslinking between Cysteines Located at Residue 101
(A) Model of the Hin tetramer undergoing clockwise subunit rotation to generate Cys101 crosslinks (see Movie S3). Cys101 residues from subunits bound to synapsed hix sites (e.g., yellow and blue subunits) are within crosslinking distance to generate hetero-diprotomer products at rotations from 0° to 80° (Figure S6C). Cys101 from subunits originally bound as dimers would not become sufficiently close to form homo-diprotomer crosslinked products (e.g., between yellow and green subunits) until almost a complete 180° rotation.
(B) Fis-activated Hin-M101C crosslinking reactions on supercoiled pRJ2330 performed as in Figure 2B.
(C) Fis-independent mutant Hin-H107Y/M101C crosslinking reactions on open-circular pRJ2330. Plus sign (+) marks background bands.
(D) Plot of the hetero-diprotomer crosslinked products from (B) and (C).
(E) Crosslinking of Hin-H107Y/M101C on an equal mixture of 3′-32P-labeled 112 bp and unlabeled 40 bp hix fragments. Crosslinking was for 20 s with BMOE at the indicated times after Hin addition. 112/40 bp synaptic complexes were then isolated by native PAGE, extracted, and subjected to SDS-PAGE. The homo-diprotomer control lane reaction contained only labeled 112 bp hix fragments.
(F) Plot of the hetero-diprotomer crosslinked products from (E).
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 Subunit Rotation on Positively Supercoiled DNA
(A) Agarose gel of pRJ2385 preparations: lane 1, negatively (−) supercoiled DNA extracted from cells; lane 2, relaxed DNA generated with topoisomerase I; lane 3, positively (+) supercoiled DNA generated with reverse gyrase.
(B and C) DNA cleavage and inversion reactions by Hin-wt, respectively. Fis-activated reactions on positively supercoiled pRJ2385 were for 10, 30, and 60 min; -Fis and all Hin reactions on negatively supercoiled DNA were for 10 min. Cleavage products were electrophoresed directly after proteinase K treatment, and inversion reactions were first cleaved with restriction enzymes to detect the inverted and parental products (Johnson and Bruist, 1989).
(D and E) Fis-activated Hin-Q134C crosslinking reactions on positively and negatively supercoiled pRJ2385, respectively.
(F) Plots of the hetero-diprotomer crosslinked products from (D) and (E).
(G and H) Fis-activated Hin-S94C crosslinking reactions on positively and negatively supercoiled pRJ2385, respectively.
(I) Plots of the hetero-diprotomer crosslinked products from (G) and (H).
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7. Figure 6 Fis-Hin Contacts Limit Processivity of Hin Subunit Rotations
(A and B) Hin crosslinking of Hin-Q134C and S94C, respectively, activated by wild-type and D20N mutant Fis. Hin and Fis were incubated with pRJ2330 for 20 min to accumulate cleaved invertasomes and then subjected to crosslinking with BMOE. The bar graphs give the means and SD of the hetero-diprotomer crosslinked products determined from five experiments.
(C) Fis-activated Hin-Q134C crosslinking reactions performed as in Figure 2B but with pRJ2123 (enhancer 6 bp closer to hixL1 than pRJ2330).
(D and E) Plots of reactions with Hin-Q134C and S94C, respectively. Data are from pRJ2123 (enhancer out-of-phase, from panel C and Figure S4B, respectively) and pRJ2330 (enhancer in-phase, from Figures 2B and 3B, respectively).
(F) Fis structure (PDB code 1F36) highlighting important residues for Hin activation and positions where cysteines were introduced for crosslinking experiments.
(G) Fis-Hin crosslinking. Fis-Q21C + Hin-WT were incubated with pRJ2372 for 20 min to accumulate cleaved invertasomes followed by crosslinking for 30 s with the cysteine-lysine heterobifunctional agents SIA (1.5 Å spacer), AMAS (4.4 Å spacer), GMBS (6.8 Å spacer), or no crosslinker. The locations of the Hin-DNA(32P) complex and Hin-DNA(32P) crosslinked with one Fis subunit are shown; the minor slowest migrating labeled species may represent two Fis subunits crosslinked to a Hin-DNA(32P) complex. Similar crosslinking reactions using AMAS were performed on pRJ2372 (enhancer in-phase) and pRJ2118 (enhancer out-of-phase) in the presence of Fis-Q21C, wild-type (control), and R71C (control).
(H) Bar graph giving the mean and SD of the percent of Fis-Hin crosslinked relative to the total number of Hin-DNA(32P) complexes obtained with pRJ2372 and pRJ2118 determined from eight reactions where crosslinking times varied from 30 s to 5 min.
(I) Plot comparing Fis-activated Hin-Q134C crosslinking reactions on pRJ2340, containing the enhancer 972 and 730 bp from the hix sites (long spacing; Figure S4C), with reactions on pRJ2330 (short spacing; Figure 2B).
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
Effects of Core Nucleotide Homology and Reaction Conditions on Rotational Conformations
(A) Schematic representation of synapsis and DNA exchange between hixL(WT) and hixL(A/T) recombination sites. After a single-round DNA exchange one of the two core nucleotides cannot base pair, which prevents ligation.
(B and C) The single-round subunit exchange property of Fis-activated Hin reactions is not disturbed by the formation of mismatched DNA recombinant products. Hin crosslinking was performed on Fis-activated Hin-Q134C (B) and S94C (C) reactions using pRJ2330 [hixL(WT) × hixL(WT)] or pRJ2383 [hixL(WT) × hixL(AT)].
(D) Ethylene glycol influences the distribution of rotational conformers. Fis-activated reactions on Hin-M101C were preformed on pRJ2383 for 20 min in reaction buffer containing the following: (lanes 1 and 2) 25% ethylene glycol plus 2 mM EDTA; (lanes 3 and 4) 25% ethylene glycol plus 10 mM MgCl2; (lanes 5 and 6) part of the reaction in lanes 1 and 2 was diluted in Mg2+-free EDTA buffer to a final concentration of 5% ethylene glycol; (lanes 7 and 8) same as lanes 5 and 6 except the dilution buffer contained 10 mM MgCl2; (lanes 9 and 10) independent reactions were performed in the same manner as in lanes 7 and 8; (lanes 11 and 12) ethylene glycol (25% final concentration) was added back to part of the reaction shown in lanes 10 and 11. The reactions were quenched without (−) or after (+) BMOE crosslinking for 1 min. Note that ligation coupled with reversal of the Hin-DNA linkage, which would normally occur upon dilution of the ethylene glycol and addition of Mg2+, is inhibited because the substrate contains nonidentical core nucleotides. The ratios of crosslinked hetero-diprotomer/homo-diprotomer products averaged from two independent experiments are given below the lanes.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions