Abstract
The RecA, UmuC, and UmuD' proteins are essential for error-prone, replicative bypass of DNA lesions. Normally, RecA protein mediates homologous pairing of DNA. We show that purified Umu(D')2C blocks this recombination function. Biosensor measurements establish that the mutagenic complex binds to the RecA nucleoprotein filament with a stoichiometry of one Umu(D')2C complex for every two RecA monomers. Furthermore, Umu(D')2C competitively inhibits LexA repressor cleavage but not ATPase activity, implying that Umu(D')2C binds in or proximal to the helical groove of the RecA nucleoprotein filament. This binding reduces joint molecule formation and even more severely impedes DNA heteroduplex formation by RecA protein, ultimately blocking all DNA pairing activity and thereby abridging participation in recombination function. Thus, Umu(D')2C restricts the activities of the RecA nucleoprotein filament and presumably, in this manner, recruits it for mutagenic repair function. This modulation by Umu(D')2C is envisioned as a key event in the transition from a normal mode of genomic maintenance by "error-free" recombinational repair, to one of "error-prone" DNA replication.
Highlights
From the ‡Division of Biological Sciences, Sections of Microbiology and of Molecular and Cellular Biology, University of California, Davis, California 95616-8665, the ¶Department of Biological Sciences, Hedco Molecular Biology Laboratories, University of Southern California, Los Angeles, California 90089-1340, and the ʈSection on DNA Replication, Repair and Mutagenesis, NICHD, National Institutes of Health, Bethesda, Maryland 20892-2725
Given that the molecular mass of the Umu(DЈ)2C complex (ϳ 71.4 kDa) is 1.8-fold greater than the RecA protein (ϳ 38.7 kDa), if each mutagenic complex interacted with a single RecA monomer, the refractive index increase upon saturation by Umu(DЈ)2C would be 1.8 times that of the RecA nucleoprotein filament; if it interacted with only the tip of each filament, the increase would be negligible
We have established that the mature mutagenesis complex Umu(DЈ)2C binds directly to the activated form of RecA protein, the nucleoprotein filament that assembles on ssDNA, and that the binding site of this complex is likely to be the deep groove between turns of the RecA nucleoprotein helix
Summary
From the ‡Division of Biological Sciences, Sections of Microbiology and of Molecular and Cellular Biology, University of California, Davis, California 95616-8665, the ¶Department of Biological Sciences, Hedco Molecular Biology Laboratories, University of Southern California, Los Angeles, California 90089-1340, and the ʈSection on DNA Replication, Repair and Mutagenesis, NICHD, National Institutes of Health, Bethesda, Maryland 20892-2725. Umu(DЈ)2C affects the ssDNA-dependent ATP hydrolysis activity of the RecA protein minimally.2 Collectively, these data argue that a specific interaction between the Umu(DЈ)2C complex and the RecA protein nucleoprotein filament is responsible for abrogating recombination function and, providing DNA polymerase III
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