Characterisation of the catalytically active form of RecG helicase

Replication of DNA is fraught with difficulty and chromosomes contain many lesions which may block movement of the replicative machinery. However, sev eral mechanisms to overcome such problems are beginning to emerge from stud ies with Escherichia coli. An important enzyme in one or more of these mech anisms is the RecG helicase, which may target stalled replication forks to generate a four-stranded (Holliday) junction, thus facilitating repair and/ or bypass of the original lesion. To begin to understand how RecG might cat alyse regression of fork structures, we have analysed what the catalyticall y active form of the enzyme may be. We have found that RecG exists as a mon omer in solution as measured by gel filtration but when bound to junction D NA the enzyme forms two distinct protein-DNA complexes that contain one and two protein molecules, However, mutant inhibition studies failed to provid e any evidence that RecG acts as a multimer in vitro. Additionally, there w as no evidence for cooperativity in the junction DNA-stimulated hydrolysis of ATP. These data suggest that RecG functions as a monomer to unwind junct ion DNA, which supports an 'inchworm' rather than an 'active rolling' mecha nism of DNA unwinding. The observed in vivo inhibition of wild-type RecG by mutant forms of the enzyme was attributed to occlusion of the DNA target a nd correlates with the very low abundance of replication forks within an E. coli cell, even during rapid growth.