Generation of double Holliday junction DNAs and their dissolution/resolution within a chromatin context

Abstract

Significance We devised a method involving DNAzyme self-cleavage for the preparation of DNA molecules containing double Holliday junctions (dHJs) that are separated by 746 bp. The DNAs can be prepared in large amounts suitable for in vitro analysis of enzymes required for the processing of recombination intermediates. We show that the dHJ DNA is dissolved efficiently by the BLM–TopIIIα–RMI1–RMI2 (BTRR) complex to produce noncrossover products, whereas nucleolytic resolution by GEN or SMX results in both crossover and noncrossover products. Following nucleosome assembly, the chromatinized dHJ structures are preferentially dissolved by BTRR. These new Holliday junction substrates will provide a valuable resource for the mechanistic analyses of the way in which recombination intermediates are processed.