Facilitated Loading of RecA Protein Is Essential to Recombination by RecBCD Enzyme

Deana A Arnold,Stephen C Kowalczykowski

doi:10.1074/jbc.275.16.12261

Abstract

Although the RecB(2109)CD enzyme retains most of the biochemical functions associated with the wild-type RecBCD enzyme, it is completely defective for genetic recombination. Here, we demonstrate that the mutant enzyme exhibits an aberrant double-stranded DNA exonuclease activity, intrinsically producing a 3'-terminal single-stranded DNA overhang that is an ideal substrate for RecA protein-promoted strand invasion. Thus, the mutant enzyme constitutively processes double-stranded DNA in the same manner as the chi-modified wild-type RecBCD enzyme. However, we further show that the RecB(2109)CD enzyme is unable to coordinate the loading of RecA protein onto the single-stranded DNA produced, and we conclude that this inability results in the recombination-defective phenotype of the recB2109 allele. Our findings argue that the facilitated loading of RecA protein by the chi-activated RecBCD enzyme is essential for RecBCD-mediated homologous recombination in vivo.

Highlights

The RecBCD enzyme is a multifunctional protein complex essential to the main pathway of homologous recombination in Escherichia coli [1, 2]
RecB2109CD Enzyme Degrades the 5Ј-Terminal Strand More Extensively Than the 3Ј-Terminal Strand—The dsDNA exonuclease activity of the wild-type RecBCD enzyme is asymmetric; the 3Ј to 5Ј nuclease activity is more vigorous than the 5Ј to 3Ј nuclease activity, which results in a more extensive degradation of the 3Ј-terminal strand at the entry site of the enzyme [11]
The ability of RecBCD enzyme to initiate recombination is attributed to two consequences of its interaction with the ␹ recombination hot spot, nuclease modification and facilitated RecA protein loading (Fig. 3)

Summary

Introduction

The RecBCD enzyme is a multifunctional protein complex essential to the main pathway of homologous recombination in Escherichia coli [1, 2]. As expected, when the 5Ј-end-labeled substrate is processed by wild-type RecBCD enzyme, there is sufficient degradation (3Ј to 5Ј) of the top strand to limit detection of a full-length ssDNA over this range of magnesium acetate concentrations (Fig. 1B, lanes 15–19).

Results

Conclusion