Figure 5.
Comparison of DNA binding modes of nucleases. (A) Superposition of the SLX1–SLX4SAP+CCD-DNA complex structure (yellow) with that of the R. Eco29KI-DNA complex (PDB: 3MX4, cyan) shows the difference of DNA conformation and positioning of the DNA cleavage sites. The structures were aligned via their Uri nuclease domains, and SLX4 is not shown for viewing clarity. The DNA bound to R. Eco29kI is colored green, and that of SLX1 is shown in orange. Active site residues in both enzymes, and nearby DNA backbone phosphate groups are shown in a stick model, colored magenta for the SLX1 complex, and cyan and red for that of the R. Eco29KI complex. (B) A close-up view of the superimposed active sites. (C) Superposition of the 5′-flap DNA substrate of FEN1 (green; PDB ID: 5KSE) with the 1-nt 5′-flap DNA of the SLX1–SLX4 complex, which is shown in a semi-transparent electrostatic potential surface representation. Three DNA basepairs next to the flap junction (–1 to –3 position) were used for alignment. The superposition shows notable differences of DNA structure at the pre-nick end of DNA, and the post-nick portion of FEN1 DNA is nearly perpendicular to the SLX1–SLX4 DNA. The 5′-flap of FEN1 DNA is next to an open surface area, indicated by a dashed-line circle, enriched with positively charged residues. (D) Sequencing gel (left panel) shows that SLX1–SLX4 cuts 1-nt 5′-flap DNA mainly at the –5 position, in contrast to the –3 position with DNA having a longer 5′-flap. The DNA marker for the 1-nt 5′-flap DNA substrate is a mixture of synthetic oligos of difference length, all with a 3′-Cy3 label, starting at the –2 nucleotide following the sequence of the flap strand displayed in the right panel. The marker for the 5′-flap DNA substrates is the same as that used in Figure 4C, as also displayed in the right panel.