Holliday junction resolving enzymes of archaeal viruses SIRV1 and SIRV2

In the final stages of genetic recombination, Holliday junction resolving e nzymes transform the four-way DNA intermediate into two duplex DNA molecule s by introducing pairs of staggered nicks flanking the junction. This funda mental process is apparently common to cells from all three domains of life . Two cellular resolving enzymes from extremely thermophilic representative s of both kingdoms of the domain Archaea, the euryarchaeon Pyrococcus furio sus and the crenarchaeon Sulfolobus solfataricus, have been described recen tly. Here we report for the first time the isolation, purification and char acterization of Holliday junction cleaving enzymes (Hjc) from two archaeal viruses. Both viruses, SIRV1 and SIRV2, infect Sulfolobus islandicus. Their Hjcs both consist of 121 amino acid residues (aa) differing only by 18 aa. Both proteins bind selectively to synthetic Holliday-structure analogues w ith an apparent dissociation constant of 25 nM. In the presence of Mg2+ the enzymes produce identical cleavage patterns near the junction. While S. is landicus shows optimal growth at about 80 degreesC, the nucleolytic activit ies of recombinant SIRV2 Hjc was highest between 45 degreesC and 70 degrees C. Based on their specificity for four-way DNA structures the enzymes may p lay a general role in genetic recombination, DNA repair and the resolution of replicative intermediates. (C) 2001 Academic Press.