Structure of a Holliday junction complex reveals mechanisms governing a highly regulated DNA transaction
- Brown University, United States
- Brandeis University, United States
- Janelia Research Campus, Howard Hughes Medical Institute, United States
- University of Pennsylvania, United States
Figures
Figure 1
Integration and excision of the λ viral chromosome into and out of the bacterial host chromosome is highly directional and tightly regulated.
(A) Formation of the integrated prophage. In those infected cells where the decision has been made not to replicate the viral DNA, the circularized supercoiled viral DNA (black lines) is inserted …
Figure 2
Patterns of specific Int bridges between core- and arm-type DNA binding sites.
(A) The amino-terminal domain (NTD) of Int (yellow) binds to one of the five arm-type DNA sites (green) via a helix-turn-helix recognition motif. It is joined via a flexible 10 residue linker region …
Figure 3
Isolation and single particle electron cryo-microscopy of the Holliday junction recombination intermediate.
(A) Native polyacrylamide gel electrophorograms of the recombination reactions prior to and after sucrose gradient purification. Recombination reactions between attL and attR partners designed to …
Figure 4
Electron cryo-microscopy refinement and 3D classification.
(A) Different stages of refinement and classification using FREALIGN (Lyumkis et al., 2013). Reconstructions at each stage are shown. The initial map was generated from 2D class averages (Figure 3C) …
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Figure 4—source data 1
FSC data in Excel format.
FSC, randomized FSC, and true FSC are tabulated as a function of resolution.
- https://doi.org/10.7554/eLife.14313.007
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Figure 4—source data 2
FSC data in csv format.
FSC, randomized FSC, and true FSC are tabulated as a function of resolution.
- https://doi.org/10.7554/eLife.14313.008
Figure 5
Model and reconstructed density for the λ excision complex.
(A) View from the 'top' of the complex, illustrating the IHF-mediated bends at H2 and H' and the close, parallel trajectory of the P and P' arms. Unmodeled density that could be occupied by Int-C …
Figure 6
The λ excision complex structure.
(A) View towards the attR-derived half of the complex, where the path of the P arm can be seen. A sharp IHF (yellow) directed bend at the H2 site, combined with the cooperative binding and bending …
Figure 7
Model for isomerization of HJ intermediate.
The core HJ complex is fit to the reconstructed density, based on structure 1Z1G (Protein Data Bank) (Biswas et al., 2005). (A) The HJ isomer corresponding to a top-strand cleavage configuration, …
Figure 8
Model of the λ integration complex.
(A) View towards the B'–C face of the complex, where the trajectory of the P arm can be seen. IHF bending at the H2 site, combined with an A-tract bend at the Fis site and additional bending …
Figure 9
Schematic of the λ integration (Ia–Id) and excision (Ea–Ec) pathways, based on the structural models presented here.
(Ia) In the presence of Int and IHF (but not Xis), an integration complex is assembled on supercoiled attP. We suggest that the Int-B CTD (blue) transiently associates with the Int-C' and Int-B' …
Videos
Video 1
The bacteriophage λ excisive HJ intermediate.
Multiple views of the excisive complex are shown. The complex is stripped of all proteins to illustrate the path of the DNA arms as they loop around the 4-way junction. IHF, Xis, and Int subunits …
Video 2
Isomerization model for the λ HJ intermediate.
The core HJ is shown switching between the isomers described in Figure 7. This type of isomerization could occur within the core of the recombination complex, without large changes in positions of …
Tables
Table 1
FRET vs. EM model distances in the λ excisive recombination complex. R(FRET) values were determined by Seah et al. (2014) using in-gel fluorescence measurements for dye pairs whose positions were …
| Position i | Position j | R (FRET) (Å) | R (FRET) Model) (Å) | R (EM Model) (Å) |
|---|---|---|---|---|
| 7 [P+50T] | 6 [P+17B] | 56 | 57 | 50.0 |
| 4 [P-58B] | 5 [P-15T] | 70 | 72 | 70.6 |
| 5 [P-15T] | 12 [B+17B] | 76 | 77 | 78.8 |
| 4 [P-58B] | 6 [P+17B] | 76 | 70 | 67.9 |
| 5 [P-15T] | 6 [P+17B] | 89 | 80 | 81.1 |
| 6 [P+17B] | 11 [B-15T] | 93 | 81 | 85.3 |
| 7 [P+50T] | 4 [P-58B] | 99 | 125 | 108.9 |
| 11 [B-15T] | 12 [B+17B] | 103 | 86 | 82.5 |
| 3 [P-118T] | 12 [B+17B] | 80 | 69 | 64.1 |
| 3 [P-118T] | 5 [P-15T] | 82 | 77 | 95.3 |
| 3 [P-118T] | 11 [B-15T] | 99 | 108 | 100.0 |
| 8 [P+79B] | 3 [P-118T] | 56 | 56 | 41.4 |
| 8 [P+79B] | 11 [B-15T] | 74 | 65 | 75.2 |
| 8 [P+79B] | 5 [P-15T] | 96 | 109 | 81.3 |
| 8 [P+79B] | 6 [P+17B] | 96 | 107 | 85.4 |
| 8 [P+79B] | 4 [P-58B] | 108 | 158 | 124.8 |
| 8 [P+79B] | 12 [B+17B] | 116 | 73 | 68.2 |
