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Fig. 1. Structure of the χ:ψ heterodimer (A) Ribbon diagram of the χ:ψ heterodimer crystal structure. ψ is colored cyan and sits on top of the χ subunit. χ is colored green except for the stretch of residues that reside in the ψ binding site which have been colored red. (B) An enlarged view of the contiguous loop region of χ and how it interacts within the cleft of ψ. This loop region has high sequence similarity with a DNA-dependent DNA polymerase from the bacteriophage PRD1. (C) A rotated view of the surface of ψ is shown. ψ has been rotate to show the cleft between α1 and α4 that makes up the χ binding surface. In green are residues 61-66 of χ. The side chain of Phe 64 of χ inserts itself in to a conserved hydrophobic pocket consisting of Val 57, Leu 121, Trp 122 and Ile 125. (D) A schematic diagram of the χ:ψ heterodimer. |
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Fig. 2. Structural comparisons of χ and ψ with other proteins (A) A side-by-side comparison of ψ with the mismatch specific DNA uracyl glycosylase MUG [45]. Similar structural features are colored yellow. (B) A side-by-side comparison of χ DEAD box helicase PcrA [46]. |
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Fig. 3. Sequence conservation in χ and ψ The conservation score using the BLOSUM62 substitution matrix, (see Materials and Methods) for each residue in χ and ψ was calculated for the 12 pairs of sequences shown in Table 2. The surfaces of χ and ψ shown in this figure are colored according to this conservation score. To the right, the binding surfaces of both proteins are shown. Both binding surfaces have been conserved in each protein. On ψ, little surface conservation is observed outside of the χ binding surface. In χ , a large amount of surface area is conserved distal to the ψ binding site. This area is proposed to bind to single stranded DNA binding protein (SSB). |
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Fig. 4. Potential χ:SSB interaction A region of χ with high sequence conservation is shown (middle). This surface is suggested to bind to the negatively charged C-terminus tail of SSB. Absolutely conserved and positively charged residues located within this region are shown on the left in a ribbon diagram in the same orientation. A schematic drawing of the inferred interaction between χ and the C-terminus consensus sequence of SSB is shown on the left. |
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Fig. 5. Conservation of sequences in the N-terminal segment of ψ An alignment of the first 26 residues of ψ, from the list of sequences given in Table 2, is shown. The alignment is colored according to the degree of sequence conservation. These 26 residues are disordered in the crystal structure of the χ:ψ complex yet a high amount of conservation is observed. It is proposed that the this linker binds to the clamp loader complex tethering the χ:ψ heterodimer to the complex. |
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Fig. 6. Potential clamp loader: ψ interaction (A) Two views of the E. coli clamp loader complex are shown [17]. An exposed hydrophobic region of the γ subunit that is highly conserved but is not involved in nucleotide binding or inter-subunit interactions is indicated as a potential binding site for the N-terminal disordered region of ψ. (B) On the left are space filling structures of the E. coli clamp loader and χ:ψ heterodimer colored by different subunits: δ (magenta), δ' (orange),γ1 (green), γ2 (red), γ3 (blue), ψ (cyan), and χ (dark green). A schematic diagram showing a possible mode of interaction between the χ:ψ heterodimer and the clamp-loader complex is shown on the right. The χ:ψ heterodimer is believed to sit in the gap between δ and δ' while the N-terminus of ψ interacts with the proposed binding region of γ inside the C-terminal collar of the clamp loader complex. |
