Recombinational branch migration by the RadA/Sms paralog of RecA in Escherichia coli

Cooper, Deani L.; Lovett, Susan T.

doi:10.7554/elife.10807

Cited by 45 publications

(80 citation statements)

References 77 publications

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“…6A and Table 3). In E. coli, it has been shown that dsDNA and ssDNA stimulate the ATPase activity of RadA at the same level (23). Our data suggest that dsDNA and ssDNA have different actions on the ATPase activity of RadA.…”

mentioning

confidence: 51%

“…3, A and B). Interestingly, it has been shown that E. coli RadA requires ADP for DNA-binding activity, suggesting the ATPase domain has an important role for DNA binding (23). Further biochemical analysis and structural information about full-length RadA and its DNA complex are necessary for a complete understanding of how the rLonC and ATPase domains coordinate to regulate DNA-binding activity.…”

Section: Discussionmentioning

confidence: 99%

“…Genetic studies have shown that radA is involved in DNA repair and genetic recombination in various organisms, such as Escherichia coli, Bacillus subtilis, and Deinococcus radiodurans (14)(15)(16)(17)(18)(19)(20)(21)(22). Recently, a biochemical characterization of E. coli RadA protein has shown that RadA stimulates RecA-catalyzed strand exchange in vitro (23). RadA is a multidomain protein consisting of three putative functional regions (Fig.…”

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confidence: 99%

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The Lon protease-like domain in the bacterial RecA paralog RadA is required for DNA binding and repair

Inoue

Fukui

Fujii

et al. 2017

Journal of Biological Chemistry

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Homologous recombination (HR) plays an essential role in the maintenance of genome integrity. RecA/Rad51 paralogs have been recognized as an important factor of HR. Among them, only one bacterial RecA/Rad51 paralog, RadA, is involved in HR as an accessory factor of RecA recombinase. RadA has a unique Lon protease-like domain (LonC) at its C terminus, in addition to a RecA-like ATPase domain. Unlike Lon protease, RadA's LonC domain does not show protease activity but is still essential for RadA-mediated DNA repair. Reconciling these two facts has been difficult because RadA's tertiary structure and molecular function are unknown. Here, we describe the hexameric ring structure of RadA's LonC domain, as determined by X-ray crystallography. The structure revealed the two positively charged regions unique to the LonC domain of RadA are located at the intersubunit cleft and the central hole of a hexameric ring. Surprisingly, a functional domain analysis demonstrated the LonC domain of RadA binds DNA, with site-directed mutagenesis showing that the two positively charged regions are critical for this DNA-binding activity. Interestingly, only the intersubunit cleft was required for the DNA-dependent stimulation of ATPase activity of RadA, and at least the central hole was essential for DNA repair function. Our data provide the structural and functional features of the LonC domain and their function in RadA-mediated DNA repair.

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confidence: 51%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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The Lon protease-like domain in the bacterial RecA paralog RadA is required for DNA binding and repair

Inoue

Fukui

Fujii

et al. 2017

Journal of Biological Chemistry

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“…The RADβ class is a divergent class of proteins that genetically function with RAD51 and are collectively known as the RAD51 paralogs. These paralogs are RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3 proteins in mammals, Rad55 and Rad57 as well as Csm2 and Psy3 in fungi, and RadB in archaea; recently, RadA of E. coli (not to be confused with RadA of Archaea), which is a member of this RADβ class, was shown to be a novel branch migration enzyme [7]. Many of the eukaryotic paralogs form various sub-complexes [8].…”

Section: Reca: a Molecular Search Enginementioning

confidence: 99%

RecA: Regulation and Mechanism of a Molecular Search Engine

Bell

Kowalczykowski

2016

Trends in Biochemical Sciences

185

164

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Homologous recombination maintains genomic integrity by repairing broken chromosomes. The broken chromosome is partially resected to produce single-stranded DNA that is used to search for homologous dsDNA. This homology-driven ‘search and rescue’ is catalyzed by a class of DNA strand exchange proteins that are defined in relation to Escherichia coli RecA, which forms a filament on single-stranded DNA. Here, we review the regulation of RecA filament assembly and the mechanism by which RecA quickly and efficiently searches for and identifies a unique homologous sequence amongst a vast excess of heterologous DNA. Given that RecA is the prototypic DNA strand exchange protein, its behavior affords insight into the actions of eukaryotic RAD51 orthologs and their regulators, BRCA2 and other tumor suppressors.

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“…The ATPase activity of RadD was necessary for this effect, with an ATPase-dead RadD displaying a dominant negative effect in response to UV. This role also showed overlap with the functions of RadA, a RecA paralogue involved in homologous recombination (22,23), and RecG, a superfamily 2 helicase involved in stabilizing recombination intermediates and preventing origin-independent replication (24,25).…”

Section: Discussionmentioning

confidence: 97%

Escherichia coli RadD Protein Functionally Interacts with the Single-stranded DNA-binding Protein

Chen

Byrne-Nash

Cox

2016

Journal of Biological Chemistry

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The bacterial single-stranded DNA binding protein (SSB) acts as an organizer of DNA repair complexes. The radD gene was recently identified as having an unspecified role in repair of radiation damage and, more specifically, DNA double-strand breaks. Purified RadD protein displays a DNA-independent ATPase activity. However, ATP hydrolytic rates are stimulated by SSB through its C terminus. The RadD and SSB proteins also directly interact in vivo in a yeast two-hybrid assay and in vitro through ammonium sulfate co-precipitation. Therefore, it is likely that the repair function of RadD is mediated through interaction with SSB at the site of damage.In Escherichia coli the single-stranded DNA-binding protein (SSB) 2 functions as a molecular organizer of a variety of DNA replication and repair functions (1). Nearly a score of different proteins directly interact with the C-terminal eight amino acids of SSB (1-9), and newly discovered members of the SSB interactome are being described regularly. In some cases the activity of the interacting protein is stimulated by SSB. All of the proteins that interact with SSB to date have a role in some aspect of DNA metabolism. Therefore, an interaction with the C terminus of SSB provides an increasingly reliable indicator of DNA metabolic function.The radD gene (formerly yejH) was recently implicated in the repair of DNA double-strand breaks after radiation or chemical damage (10,11). The peptide sequence of the RadD protein includes all seven of the motifs associated with a superfamily 2 (SF2) helicase (10). Although there is little homology outside of these regions, the helicase motifs align well to other E. coli SF2 helicases including RecG and RecQ. RadD also contains a putative zinc finger motif in the C terminus that could aid in binding to DNA substrates. Both the ATPase and zinc binding domains are necessary for the rescue of DNA repair activities of radD deletion strains in vivo (10).Here, we present the first biochemical characterization of the RadD protein. Although helicase activity was not observed, RadD hydrolyzes ATP in the absence of DNA. This activity is stimulated by the SSB protein, specifically the C-terminal amino acid residues of SSB. The RadD and SSB proteins interact in vivo and directly in vitro. The previous study and this work, taken together, suggest that SSB likely recruits and activates RadD in the cell during DNA repair. ResultsRationale-Based on the sequence similarity of RadD to known DNA metabolism proteins and a recent study implicating RadD in the repair of double-strand DNA breaks (10), we natively purified the RadD protein and screened for likely activities and interactions. A number of assays for helicase activity, including a variety of both DNA and RNA substrates, produced no positive results and thus are not reported. Presented here are results that demonstrate DNA-independent ATPase activity for RadD as well as an interaction with the SSB C terminus.RadD Protein Has a DNA-independent ATPase Activity-As a predicted SF2 helicase (10), Rad...

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Recombinational branch migration by the RadA/Sms paralog of RecA in Escherichia coli

Cited by 45 publications

References 77 publications

The Lon protease-like domain in the bacterial RecA paralog RadA is required for DNA binding and repair

The Lon protease-like domain in the bacterial RecA paralog RadA is required for DNA binding and repair

RecA: Regulation and Mechanism of a Molecular Search Engine

Escherichia coli RadD Protein Functionally Interacts with the Single-stranded DNA-binding Protein

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