The architecture of the human Rad54-DNA complex provides evidence for protein translocation along DNA - PubMed (original) (raw)

The architecture of the human Rad54-DNA complex provides evidence for protein translocation along DNA

D Ristic et al. Proc Natl Acad Sci U S A. 2001.

Abstract

Proper maintenance and duplication of the genome require accurate recombination between homologous DNA molecules. In eukaryotic cells, the Rad51 protein mediates pairing between homologous DNA molecules. This reaction is assisted by the Rad54 protein. To gain insight into how Rad54 functions, we studied the interaction of the human Rad54 (hRad54) protein with double-stranded DNA. We have recently shown that binding of hRad54 to DNA induces a change in DNA topology. To determine whether this change was caused by a protein-constrained change in twist, a protein-constrained change in writhe, or the introduction of unconstrained plectonemic supercoils, we investigated the hRad54--DNA complex by scanning force microscopy. The architecture of the observed complexes suggests that movement of the hRad54 protein complex along the DNA helix generates unconstrained plectonemic supercoils. We discuss how hRad54-induced superhelical stress in the target DNA may function to facilitate homologous DNA pairing by the hRad51 protein directly. In addition, the induction of supercoiling by hRad54 could stimulate recombination indirectly by displacing histones and/or other proteins packaging the DNA into chromatin. This function of DNA translocating motors might be of general importance in chromatin metabolism.

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Figures

Figure 1

Figure 1

SFM images of nicked circular DNA molecules in the absence or presence of hRad54. The images were processed only by flattening to remove background slope and are presented as top views. All images show an area of 500 nm × 500 nm, zoomed in from 2-μm × 2-μm scans. The z dimension is indicated by color as shown on the bar at the right. (A) DNA molecules without hRad54. (B) hRad54–DNA complexes formed in the absence ATP. (C) hRad54–DNA complexes formed in the presence of ATP.

Figure 2

Figure 2

Histograms of DNA contour length measured from molecules with or without bound hRad54. hRad54–DNA complexes were formed in the presence of ATP. All DNA length measurements were from images collected from one deposition of molecules from one reaction mixture. The top left of each panel shows the number of DNA molecules measured, their average contour length, and standard deviation. (A) Contour length of DNA molecules without bound protein. (B) Contour length of DNA molecules with bound hRad54.

Figure 3

Figure 3

SFM image of hRad54–DNA complexes formed in the presence of ATP. The image is presented as line plot at a 60° viewing angle to emphasize topography. Height is indicated by color as shown on the bar at the right. One plasmid has a hRad54 complex bound at the junction of relaxed and supercoiled domains. The other plasmid has two hRad54 complexes bound.

Figure 4

Figure 4

Comparison of the size of large hRad54 complexes bound to DNA in the presence of ATP and proteins of known size. SFM image of a mixture of hRad54, RNA polymerase, and Ku70/80 bound to the different DNA substrates. Upper left, circular DNA–hRad54 complex (hRad54 monomer is 87.8 kDa); lower left, RNA polymerase (450 kDa) bound to a long linear DNA (partially shown); lower middle, three Ku70/80 heterodimers (155 kDa) bound to a short linear DNA.

Figure 5

Figure 5

Model for generation of supercoiling by hRad54 translocation along DNA. The hRad54 complex and plasmid DNA are indicated by the shaded oval and black line, respectively. (A) Movement of the hRad54 complex by tracking along the helical path of DNA is indicated by the arrows. When the complex is free to rotate around the DNA, no change in supercoiling will be induced in the plasmid DNA. (B) When the hRad54 complex tracks along the helix, while being prevented from rotating around the DNA, positive supercoils will arise ahead of the protein complex and negative supercoils behind it. These supercoils can freely distribute along the plasmid and therefore they will cancel each other out. (C) The interaction of two hRad54 complexes on a plasmid will divide the plasmid into two domains. Because the plasmid is singly nicked, one domain will contain a nick, whereas the other contains two covalently closed DNA strands. Depending on the position of the nick relative to the movement of the protein complex along the DNA, topoisomers containing either negative or positive supercoils will result after ligation of the nick.

Figure 6

Figure 6

Model for stimulation of Rad51-mediated joint molecule formation by Rad54 translocation. A chromosomal domain is indicated by the black line connecting the hatched areas. The Rad54 protein complex is represented by the shaded oval, and it is shown to interact with the Rad51 nucleoprotein filament that is assembled on the broken DNA indicated in gray (A, before hRad54 translocation). This interaction will provide the frictional torque that prevents the Rad54 complex from rotating around the DNA as it tracks along the helix. In this way, movement of the Rad54 complex along the DNA (B, after hRad54 translocation) will generate negative and positive supercoils into the domains divided by the Rad54 complex. See text for details on how this process might stimulate Rad51-mediated joint molecule formation.

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