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The blockage of replication forks can result in the disassembly of the replicative apparatus and reversal of the fork to form a DNA junction that must be processed in order for replication to restart and sister chromatids to segregate at mitosis. Fission yeast Mus81-Eme1 and budding yeast Mus81-Mms4 are endonucleases that have been implicated in the processing of aberrant DNA junctions formed at stalled replication forks. Here we have investigated the activity of purified Mus81-Eme1 and Mus81-Mms4 on substrates that resemble DNA junctions that are expected to form when a replication fork reverses. Both enzymes cleave Holliday junctions and substrates that resemble normal replication forks poorly or not at all. However, forks where the equivalents of either both the leading and lagging strands or just the lagging strand are juxtaposed at the junction point, or where either the leading or lagging strand has been unwound to produce a fork with a single-stranded tail, are cleaved well. Cleavage sites map predominantly between 3 and 6 bp 5' of the junction point. For most substrates the leading strand template is cleaved. The sole exception is a fork with a 5' single-stranded tail, which is cleaved in the lagging strand template.

Original publication

DOI

10.1074/jbc.M210006200

Type

Journal article

Journal

J Biol Chem

Publication Date

28/02/2003

Volume

278

Pages

6928 - 6935

Keywords

DNA, DNA-Binding Proteins, Dimerization, Endonucleases, Escherichia coli, Flap Endonucleases, Models, Genetic, Plasmids, Protein Binding, Saccharomyces cerevisiae Proteins, Saccharomycetales, Schizosaccharomyces, Schizosaccharomyces pombe Proteins, Time Factors, Trans-Activators