Recognition and Distortion of DNA Structure by Junction-Resolving Enzymes; Importance in Ensuring Productive Resolution.
Junction resolving enzymes catalyse the resolution of four-way (Holliday) junctions as the penultimate stage of homologous genetic recombination. To do this they must create two scissions in the DNA backbone, precisely placed symmetrically on different arms of the junction. Enzymes in this class always bind as dimers, and a bilateral cleavage event arises when each subunit cleaves one strand of the junction to which they are bound. The binding step is driven by a very high structural specificity, but these enzymes distort the very feature that they recognise upon binding and each of them constrains the junction into a different, well-defined conformation(1).
We have used 2-aminopurine (2AP) fluorescence as a reporter of the local conformation of the center of a junction upon binding of resolving enzymes: when incorporated into duplex DNA, this adenine analog exhibits a strong de-quenching of its fluorescence emission if it is unstacked. We have observed significant fluorescence increases upon binding resolving enzymes when the reporter 2AP base was adjacent to the junction center, the most extreme example being the yeast enzyme CCE1 which completely unstacks the four central basepairs(2). In order to study how this structural distortion relates to the catalytic activity, we have used an assay based on a supercoil-stabilised cruciform to assess the simutaneity or coordination of the two cleavage reactions. We find that although both cleavages are not simultaneous, they occur within the lifetime of the junction-enzyme complex and are not completely independent: the rate of cleavage of a given site is accelerated when it occurs subsequently to the initial cleavage. Moreover, using slow cutting sequences, we show that the enzyme can stabilise the cruciform structure for long times and only dissociates after the second cleavage has been introduced(3). These results taken together suggest that the structural strain imposed on the junction upon binding of the enzyme is released by the first cleavage event, which leads to a more stable junction-enzyme complex and an increased efficiency of the second cleavage step. Thus the structural manipulation of the junction is the key element to ensure its productive resolution.
Finally, we recently solved the structure of the endonuclease I of phage T7, which opens a new perspective for our study of this class of enzymes.
(1) M F White, M -J E Giraud-Panis, J R G Pohler and D M J Lilley (1997) J Molec Biol 269, 647-664.
Anne-Cecile Declais, Jonathan Fogg, Jonathan Hadden1, Maire Convery1, Simon EV Phillips1 and David MJ Lilley.
CRC Nucleic Acid Structure Research Group, Department of Biochemisty,