Analysis of conserved basic residues associated with DNA binding (Arg69) and catalysis (Lys76) by the RusA Holliday junction resolvase

Holliday junctions are key intermediates in both homologous recombination a nd DNA repair, and are also formed from replication forks stalled at lesion s in the template strands. Their resolution is critical for chromosome segr egation and cell viability, and is mediated by a class of small, homodimeri c endonucleases that bind the structure and cleave the DNA. All the enzymes studied require divalent metal ions for strand cleavage and their active c entres are characterised by conserved aspartate/glutamate residues that pro vide ligands for metal binding. Sequence alignments reveal that they also c ontain a number of conserved basic residues. We used site-directed mutagene sis to investigate such residues in the RusA resolvase. RusA is a 120 amino acid residue polypeptide that can be activated in Escherichia coli to prom ote recombination and repair in the absence of the Ruv proteins. The RuvA, RuvB and RuvC proteins form a complex on Holliday junction DNA that drives coupled branch migration (RuvAB) and resolution (RuvC) reactions. In contra st to RuvC, the RusA resolvase does not interact directly with a branch mig ration motor, which simplifies analysis of its resolution activity. Catalys is depends on three highly conserved acidic residues (Asp70, Asp72 and Asp9 1) that define the catalytic centre. We show that Lys76, which is invariant in RusA sequences, is essential for catalysis, but not for DNA binding, an d that an invariant asparagine residue (Asn73) is required for optimal acti vity. Analysis of DNA binding revealed that RusA may interact with one face of an open junction before manipulating its conformation in the presence o f Mg2+ as part of the catalytic process. A well-conserved arginine residue (Arg69) is linked with this critical stage. These findings provide the firs t insights into the roles played by basic residues in DNA binding and catal ysis by a Holliday junction resolvase. (C) 2000 Academic Press.