A Single Mutation, RecBD1080A, Eliminates RecA Protein Loading but Not Chi Recognition by RecBCD Enzyme

Daniel G Anderson,Jason J Churchill,Stephen C Kowalczykowski

doi:10.1074/jbc.274.38.27139

Daniel G Anderson, Jason J Churchill + Show 1 more

Open Access

https://doi.org/10.1074/jbc.274.38.27139

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Abstract

Homologous recombination and double-stranded DNA break repair in Escherichia coli are initiated by the multifunctional RecBCD enzyme. After binding to a double-stranded DNA end, the RecBCD enzyme unwinds and degrades the DNA processively. This processing is regulated by the recombination hot spot, Chi (chi: 5'-GCTGGTGG-3'), which induces a switch in the polarity of DNA degradation and activates RecBCD enzyme to coordinate the loading of the DNA strand exchange protein, RecA, onto the single-stranded DNA products of unwinding. Recently, a single mutation in RecB, Asp-1080 --> Ala, was shown to create an enzyme (RecB(D1080A)CD) that is a processive helicase but not a nuclease. Here we show that the RecB(D1080A)CD enzyme is also unable to coordinate the loading of the RecA protein, regardless of whether chi sites are present in the DNA. However, the RecB(D1080A)CD enzyme does respond to chi sites by inactivating in a chi-dependent manner. These data define a locus of the RecBCD enzyme that is essential not only for nuclease function but also for the coordination of RecA protein loading.

Highlights

In Escherichia coli, the early steps of homologous recombination and dsDNA1 break repair are catalyzed by the RecBCD enzyme and RecA protein [1]
DNA processing by the RecBCD enzyme is regulated by an eight-base DNA element, Chi (␹; 5Ј-GCTGGTGG-3Ј) [12,13,14,15]. ␹ was originally identified in vivo as a recombination hot spot that stimulates homologous recombination about 10-fold in its vicinity
The mechanism by which RecA protein is loaded onto ssDNA by RecBCD enzyme remains unclear

Summary

The fact that RecBC enzyme has little nuclease activity

RecBD1080ACD Enzyme Cannot Load RecA Protein indicates that the RecD subunit plays an essential role in DNA degradation. A single point mutation in the putative Mg2ϩ binding site of the RecB subunit, Asp-1080 3 Ala, creates a holoenzyme (RecBD1080ACD) that behaves much like the RecBC enzyme; it is a processive helicase with no measurable nuclease activity. We show that despite being an efficient helicase, the RecBD1080ACD enzyme is unable to load RecA protein This inability to load RecA protein is independent of whether the processed DNA contains ␹ sites. The RecBD1080ACD enzyme does not load RecA protein in response to ␹, it can still recognize ␹: ␹-containing DNA is processed at a slower rate than DNA without ␹ These data provide the first insight into the domain that is responsible for either 1) transmitting the ␹ recognition event into the enzymatic alterations that are necessary for proper RecBCD enzyme function or 2) RecA protein loading

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