Figure 1.

DNA-protein interactions of endonucleases and their in vitro cleavage of distinct DNA substrates. (A) Diagram of wild-type I-CreI homodimer in complex with its natural DNA recognition sequence. One I-CreI monomer is shown in white, the other (I-CreI’) in grey. DNA sequence is indicated, with four base-pair center sequence shown in bold. Direct hydrogen bonds between I-CreI and DNA are shown as black lines. Sites of phosphodiester bond cleavage and the resulting 4 bp 3^′ overhangs are indicated by a line. A likely unfavorable electrostatic interaction between E80 and a backbone phosphate is indicated by a small arrow. (B) Predicted interactions between rationally designed PB1 endonuclease and the RS_GTAC DNA site. The two monomers (PB1 and PB1^′) and DNA interactions are as indicated in (A), except amino acids that deviate from I-CreI and I-CreI’ hydrogen bonds (or a hydrophobic interaction, C33) and are predicted to contribute to altered DNA-cleavage specificity are indicated with dashed lines. PB1+ endonuclease contains a mutation (E80 to Q80) predicted to eliminate the unfavorable interaction mentioned in (A). (C) Cleavage of DNA by native and rationally designed endonucleases. I-CreI, PBI, and PB1+ endonucleases were incubated with three distinct linearized DNA substrates (sequence indicated above its respective set of digests). Sequence differences between I-CreI (wild-type) and the two PB1 recognition sites highlighted in grey. DNA for PB1_TAGA (center) and PB1_GTAC (right) differ from each other by the 4 bp center sequence (subscript). Digests were conducted with 0, 0.007, 0.015, 0.031, 0.062, 0.125, 0.25, 0.5, 1, 2 μM endonuclease.